Aminoglycosides and uses thereof in the treatment of genetic disorders

ABSTRACT

A new class of paromomycin-derived aminoglycosides, which exhibit efficient stop-codon mutation suppression activity, low toxicity and high selectivity towards eukaryotic cells are provided. Also provided are chemical and chemo-enzymatic processes of preparing these paromomycin-derived aminoglycosides and intermediates thereof, as well as pharmaceutical compositions containing the same, and uses thereof in the treatment of genetic disorders.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/285,299 filed Oct. 1, 2008, which is a Continuation-In-Part of PCTPatent Application No. PCT/IL2007/000463 filed Apr. 10, 2007. Thecontents of the above applications are all incorporated by reference asif fully set forth herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a new class of aminoglycosides and touses thereof in the treatment of genetic disorders.

Many human genetic disorders result from nonsense mutations, where oneof the three stop codons (UAA, UAG or UGA) replaces an amino acid-codingcodon, leading to premature termination of the translation andeventually to truncated inactive proteins. Currently, hundreds of suchnonsense mutations are known, and several were shown to account forcertain cases of fatal diseases, including cystic fibrosis (CF),Duchenne muscular dystrophy (DMD), ataxia-telangiectasia, Hurlersyndrome, hemophilia A, hemophilia B, Tay-Sachs, and more [1, 2]. Formany of those diseases there is presently no effective treatment, andalthough gene therapy seems like a potential possible solution forgenetic disorders, there are still many critical difficulties to besolved before this technique could be used in humans.

During the last several years, it has been shown that aminoglycosidescould have therapeutic value in the treatment of several geneticdiseases because of their ability to induce ribosomes to read-throughstop codon mutations, generating full-length proteins from part of themRNA molecules [3-6].

Aminoglycosides are highly potent, broad-spectrum antibiotics commonlyused for the treatment of life-threatening infections [7, 8]. The2-deoxystreptamine (2-DOS) aminoglycosides antibiotics, shown inbackground art FIG. 1 [9], selectively target the prokaryotic ribosome,and, by binding to the decoding A-site of the 16S ribosomal RNA, lead toprotein translation inhibition and interference with the translationalfidelity [7, 10-12]. One of the most studied aminoglycosides isparomomycin (its sulfate salt known under its brand name Humatin), whichis an antimicrobial drug used against intestinal amebiasis. It wasapproved by the Drug Controller General of India as an agent againstvisceral leishmaniasis (kala azar) in India, and was granted “orphandrug” status in 2005 in the US. Paromomycin is known to inhibit proteinsynthesis by binding to the ribosomal RNA of the 16S subunit.

Several achievements in bacterial ribosome structure determination[13-17], along with crystal and NMR structures of bacterial A-siteoligonucleotide models [18-22], have provided useful information forunderstanding the decoding mechanism in prokaryote cells andunderstanding how aminoglycosides exert their deleterious misreading ofthe genetic code. During decoding, a critical step in aminoacyl-tRNAselection is based on the formation of a mini-helix between the codon ofthe mRNA and the anti-codon of the cognate aminoacyl-tRNA. In thisprocess, the conformation of the A-site is changed from an ‘off’ state,where the two conserved adenines A1492 and A1493 are folded back withinthe helix, to an ‘on’ state, where A1492 and A1493 are flipped out fromthe A-site and interact with the cognate codon-anticodon mini-helix [11,15]. This conformational change is a molecular switch that decides onthe continuation of translation in an irreversible way. The binding ofaminoglycosides such as paromomycin and gentamicin to the bacterialA-site stabilizes the ‘on’ conformation even in the absence of cognatetRNA-mRNA complex. Thus, the affinity of the A-site for a non-cognatemRNA-tRNA complex is increased upon aminoglycosides binding, preventingthe ribosome from efficiently discriminating between non-cognate andcognate complexes.

The termination of protein synthesis is signaled by the presence of astop codon in the mRNA, and is mediated by release factor proteins. Theefficiency of translation termination is usually very high, and inintact cells the misincorporation of an amino acid at a stop codon(suppression) normally occurs at a low frequency of around 10⁻⁴. Theenhancement of termination suppression by aminoglycosides in eukaryotesis thought to occur in a similar mechanism to the aminoglycosides'activity in interfering with translational fidelity during proteinsynthesis, namely the binding of certain aminoglycosides to theribosomal A-site probably induce conformational changes that stabilizenear-cognate mRNA-tRNA complexes, instead of inserting the releasefactor. Aminoglycosides suppress the various stop codons with notablydifferent efficiencies (UGA>UAG>UAA), and the suppression effectivenessis further dependent upon the identity of the fourth nucleotideimmediately downstream from the stop codon (C>U>A≧G) as well as thelocal sequence context around the stop codon [6, 23].

The fact that aminoglycosides could suppress premature nonsensemutations in mammalian cells was first demonstrated by Burke and Mogg in1985, who also noted the therapeutic potential of these drugs in thetreatment of genetic disorders [3]. The first genetic disease examinedwas cystic fibrosis (CF), the most prevalent autosomal recessivedisorder in the Caucasian population, affecting 1 in 2,500 newborns. CFis caused by mutations in the cystic fibrosis transmembrane conductanceregulator (CFTR) protein. Currently, more than 1,000 differentCF-causing mutations in the CFTR gene were identified, and 5-10% of themutations are premature stop codons. In Ashkenazi Jews, the W1282Xmutation and other nonsense mutations account for 64% of all CFTR mutantalleles [5].

The first experiments of aminoglycoside-mediated suppression of CFTRstop mutations demonstrated that premature stop mutations found in theCFTR gene could be suppressed by G-418 and gentamicin (see, backgroundart FIG. 1), as measured by the appearance of full-length, functionalCFTR in bronchial epithelial cell lines [24, 25]. Suppressionexperiments of intestinal tissues from CFTR−/− transgenic mice mutantscarrying a human CFTR-G542X transgene showed that treatment withgentamicin, and to lesser extent tobramycin, have resulted in theappearance of human CFTR protein at the glands of treated mice [26].Most importantly, clinical studies using double-blind,placebo-controlled, crossover trails have shown that gentamicin cansuppress stop mutations in affected patients, and that gentamicintreatment improved transmembrane conductance across the nasal mucosa ina group of 19 patients carrying CFTR stop mutations [27]. Other geneticdisorders for which the therapeutic potential of aminoglycosides wastested in in-vitro systems, cultured cell lines, or animal modelsinclude DMD [28], Hurler syndrome [29], nephrogenic diabetes insipidus[30], nephropathic cystinosis [31], retinitis pigmentosa [32], andataxia-telangiectasia [33].

However, one of the major limitations in using aminoglycosides aspharmaceuticals is their high toxicity towards mammals, typicallyexpressed in kidney (nephrotoxicity) and ear-associated (ototoxicity)illnesses. The origin of this toxicity is assumed to result from acombination of different factors and mechanisms such as interactionswith phospholipids, inhibition of phospholipases and the formation offree radicals [34, 35]. Although considered selective to bacterialribosomes, most aminoglycosides bind also to the eukaryotic A-site butwith lower affinities than to the bacterial A-site [36]. The inhibitionof translation in mammalian cells is also one of the possible causes forthe high toxicity of these agents. Another factor adding to theircytotoxicity is their binding to the mitochondrial 12S rRNA A-site,whose sequence is very close to the bacterial A-site [37].

Many studies have been attempted to understand and offer ways toalleviate the toxicity associated with aminoglycosides [38], includingthe use of antioxidants to reduce free radical levels [39, 40], as wellas the use of poly-L-aspartate [41, 42] and daptomycin [43, 44] toreduce the ability of aminoglycosides to interact with phospholipids.The role of megalin (a multiligand endocytic receptor which isespecially abundant in the kidney proximal tubules and the inner ear) inthe uptake of aminoglycosides has recently been demonstrated [35]. Theadministration of agonists that compete for aminoglycoside binding tomegalin also resulted in a reduction in aminoglycoside uptake andtoxicity [45]. In addition, altering the administration schedule and/orthe manner in which aminoglycosides are administered has beeninvestigated as means to reduce toxicity [46, 47].

Despite extensive efforts to reduce aminoglycoside toxicity, few resultshave matured into standard clinical practices and procedures for theadministration of aminoglycosides to suppress stop mutations, other thanchanges in the administration schedule. For example, the use ofsub-toxic doses of gentamicin in the clinical trails probably caused thereduced read-through efficiency obtained in the in-vivo experimentscompared to the in-vitro systems [48]. The aminoglycoside Geneticin®(G-418 sulfate, see, background art FIG. 1) showed the best terminationsuppression activity in in-vitro translation-transcription systems [6],however, its use as a therapeutic agent is not possible since it islethal even at very low concentrations. For example, the LD₅₀ of G-418against human fibroblast cells is 0.04 mg/ml, compared to 2.5-5.0 mg/mlfor gentamicin, neomycin and kanamycin [49].

The increased sensitivity of eukaryotic ribosomes to some aminoglycosidedrugs, such as G-418 and gentamicin, is intriguing but up to date couldnot be rationally explained because of the lack of sufficient structuraldata on their interaction with eukaryotic ribosomes. Since G-418 isextremely toxic even at very low concentrations, presently gentamicin isthe only aminoglycoside tested in various animal models and clinicaltrials. Although some studies have shown that due to their relativelylower toxicity in cultured cells, amikacin [50] and paromomycin [51] canrepresent alternatives to gentamicin for stop mutation suppressiontherapy, no clinical trials with these aminoglycosides have beenreported yet.

To date, nearly all suppression experiments have been performed withclinical, commercially available aminoglycosides [6], and no effortshave been made to optimize their activity as stop codon read-throughinducers. Currently, only a limited number of aminoglycosides, includinggentamicin, amikacin, and tobramycin, are in clinical use as antibioticsfor internal administration in humans. Among these, tobramycin do nothave stop mutations suppression activity, and gentamicin is the onlyaminoglycoside tested for stop mutations suppression activity in animalmodels and clinical trials. Recently, a set of neamine derivatives wereshown to promote read-through of the SMN protein in fibroblasts derivedfrom spinal muscular atrophy (SPA) patients; however, these compoundswere originally designed as antibiotics and no conclusions were derivedfor further improvement of the read-through activity of thesederivatives [52].

U.S. patent application Ser. No. 11/073,649, by the present assignee,which is incorporated by reference as if fully set forth herein, teachesa family of aminoglycosides, which have common structural backbonefeatures which enables these aminoglycosides to be highly potent andeffective antibiotics, while reducing or blocking antibiotic resistancethereto. The aminoglycoside derivatives taught in U.S. patentapplication Ser. No. 11/073,649, are presented as effective antibioticsagainst bacterial infections such as anthrax, and also as therapeuticagents for the treatment of genetic disorder, such as cystic fibrosis.

More specifically, the compounds taught in U.S. patent application Ser.No. 11/073,649 were designed based upon known aminoglycosidesantibiotics which exert their antibacterial activity by selectivelyrecognizing and binding to the decoding A site on the 16S subunit of thebacterial rRNA. Thus, these compounds are semi-synthetic analogs ofcurrently available aminoglycosides, in which a pre-determined positionof the aminoglycoside has been modified so as to enhance the recognitionof the phosphodiester bond of rRNA and in parallel the Asp/Glu andAsn/Gln clusters in the active site of the lethal factor (LF) andthereby exhibit enhanced anti-bacterial performance. These modificationsfurther provide the compounds with resistance to enzymatic catalysis andthus improve their bioavailability and hence anti-bacterial performance.Furthermore, the steric hindrance introduced into the designedstructures via the chemical modification of the aminoglycoside, rendersthese compounds inferior substrates for the most widely representedresistance-causing enzyme, APH(3′)-IIIa, thus preventing the developmentof resistance thereto.

The design and bifunctional activity of these structures is alsodescribed by Mariana Hainrichson et al, in Bioorganic and MedicinalChemistry 13 (2005) 5797-5807.

The compounds taught in the compounds taught in U.S. patent applicationSer. No. 11/073,649 were further found to block a premature stop codonand hence effective in treating genetic disorders. However, as detailedhereinbelow, the enhanced antibacterial activity of these compounds maybe undesirable when used to treat genetic disorders. Other modifiedaminoglycosides and structurally related antibiotics have been proposedand prepared [53-61] yet the stop-codon read-through therapeuticactivity thereof was neither described nor suggested or tested.

The desired characteristics of an effective read-through drug would beoral administration and little or no effect on bacteria. Antimicrobialactivity of read-through drug is undesirable as any unnecessary use ofantibiotics, particularly with respect to the gastrointestinal (GI)biota, due to the adverse effects caused by upsetting the GI biotaequilibrium and the emergence of resistance. In this respect, inaddition to the abovementioned limitations, the majority of clinicalaminoglycosides are greatly selective against bacterial ribosomes, anddo not exert a significant effect on cytoplasmic ribosomes of humancells.

In an effort to circumvent the abovementioned limitations, thebiopharmaceutical company PTC Therapeutics (NY, USA) is trying currentlyto discover new stop mutations suppression drugs by screening largechemical libraries for nonsense read-through activity. Using thisapproach, a new non-aminoglycoside compound, PTC124, was discovered[62].

3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid (PTC124)

The facts that PTC124 is reported to have no antibacterial activity andno reported toxicity, suggest that its mechanism of action on theribosome is different than that of the aminoglycosides. The FDA hasgranted fast track and orphan drug designations to PTC124 for thetreatment of both CF and DMD caused by nonsense mutations, and thepreliminary results of phase II clinical trails in CF and DMD patientsseem promising [63-67].

SUMMARY OF THE INVENTION

In summary, the collective data presented above suggest that systematicsearch for new aminoglycoside derivatives with improved terminationmutation suppression activity, lower toxicity to mammalian cells, andlimited or no antimicrobial activity is required to exploit the avenueof aminoglycoside derivative research to the point where they can beused clinically.

The present invention relates to a new class of paromomycin derivedaminoglycosides, which can be beneficially used in the treatment ofgenetic diseases, such as cystic fibrosis, by exhibiting high prematurestop-codon mutations read-through activity while exerting low toxicityin mammalian cells and low antimicrobial activity.

Thus, according to one aspect of the present invention there is provideda compound having a general Formula I:

or a pharmaceutically acceptable salt thereof,

wherein:

each of R₁, R₂ and R₃ is independently a monosaccharide moiety, halide,hydroxyl, amine or an oligosaccharide moiety,

X is oxygen or sulfur;

R₄ is hydrogen or (S)-4-amino-2-hydroxybutyryl (AHB);

R₅ is hydroxyl or amine;

Y is hydrogen, alkyl or aryl;

the dashed line indicates an R configuration or an S configuration;

with the proviso that the compound is not selected from the groupconsisting of

amikacin, apramycin, arbekacin, butirosin, dibekacin, fortimycin, G-418,gentamicin, hygromycin, habekacin, dibekacin, netlmicin, istamycin,isepamycin, kanamycin, lividomycin, neamine, neomycin, paromomycin,ribostamycin, sisomycin, spectinomycin, streptomycin and tobramycin.

According to some embodiments, the compound having a general Formula Iis selected from the group consisting of:

According to another aspect of the present invention there is provided apharmaceutical composition comprising a compound as described herein anda pharmaceutically acceptable carrier.

According to some embodiments, the pharmaceutical composition ispackaged in a packaging material and identified in print, in or on thepackaging material, for use in the treatment of a genetic disorder.

According to some embodiments, the pharmaceutical composition isformulated for oral administration.

According to yet another aspect of the present invention there isprovided a method of treating a genetic disorder, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound having a general Formula I, with the proviso thatthe compound is not selected from the group consisting of amikacin,apramycin, arbekacin, butirosin, dibekacin, fortimycin, G-418,gentamicin, hygromycin, habekacin, dibekacin, netlmicin, istamycin,isepamycin, kanamycin, lividomycin, neamine, neomycin, paromomycin,ribostamycin, sisomycin, spectinomycin, streptomycin and tobramycin.

According to some embodiments, the compound is administered orally.

According to still another aspect of the present invention there isprovided a use of a compound having a general Formula I in themanufacture of a medicament for treating a genetic disorder, with theproviso that the compound is not selected from the group consisting ofamikacin, apramycin, arbekacin, butirosin, dibekacin, fortimycin, G-418,gentamicin, hygromycin, habekacin, dibekacin, netlmicin, istamycin,isepamycin, kanamycin, lividomycin, neamine, neomycin, paromomycin,ribostamycin, sisomycin, spectinomycin, streptomycin and tobramycin.

According to some embodiments, the genetic disorder comprises a proteinhaving a truncation mutation.

According to some embodiments, the genetic disorder is selected from thegroup consisting of cystic fibrosis (CF), Duchenne muscular dystrophy(DMD), ataxia-telangiectasia, Hurler syndrome, hemophilia A, hemophiliaB, Usher syndrome and Tay-Sachs. Preferably, the genetic disorder isselected from the group consisting of cystic fibrosis (CF), Duchennemuscular dystrophy (DMD) and Hurler syndrome.

According to features in preferred embodiments of the inventiondescribed below, X in Formula I is oxygen.

According to some embodiments, R₅ in Formula I is hydroxyl.

According to some embodiments, Y in Formula I is hydrogen.

According to some embodiments, at least one of R₁, R₂ and R₃ in FormulaI is a monosaccharide moiety.

According to some embodiments, R₁ is the monosaccharide moiety.

According to some embodiments, R₂ and R₃ are each hydroxyl.

According to some embodiments, R₂ is the monosaccharide moiety.

According to some embodiments, R₁ and R₃ are each hydroxyl.

According to some embodiments, R₃ is the monosaccharide moiety.

According to some embodiments, R₁ and R₂ are each hydroxyl.

According to some embodiments, the monosaccharide moiety has the generalFormula II:

wherein:

the dashed line indicates an R configuration or an S configuration; andeach of R₆, R₇ and R₈ is independently selected from the groupconsisting of hydroxyl and amine.

According to some embodiments, R₇ and R₈ in Formula II are eachhydroxyl.

According to some embodiments, R₆ in Formula II is amine.

According to some embodiments, R₆ in Formula II is hydroxyl.

According to some embodiments, R₁ is amine and R₂ and R₃ are eachhydroxyl.

According to some embodiments, at least of R₁, R₂ and R₃ is anoligosaccharide moiety.

According to some embodiments, R₁ is an oligosaccharide moiety.Preferably, R₂ and R₃ are each hydroxyl.

According to some embodiments, R₂ is an oligosaccharide moiety.Preferably, R₁ and R₃ are each hydroxyl.

According to some embodiments, R₃ is an oligosaccharide moiety.Preferably, R₁ and R₂ are each hydroxyl.

According to some embodiments, the oligosaccharide moiety is adisaccharide moiety.

According to some embodiments, the disaccharide moiety has the generalFormula I*:

wherein:

the dashed line indicates an R configuration or an S configuration;

each of R*₁, R*₂ and R*₃ is independently a halide, hydroxyl, amine oris linked to the compound having general Formula I, whereas at least oneof R*₁, R*₂ and R*₃ is linked to the compound having the general FormulaI above;

X* is oxygen or sulfur;

R*₄ is hydrogen or an (S)-4-amino-2-hydroxybutyryl (AHB) moiety;

R*₅ is hydroxyl or amine; and

Y* is hydrogen, alkyl or aryl.

According to some embodiments, the oligosaccharide moiety furthercomprises a linker.

According to some embodiments, R₄ and Y are each hydrogen.

According to some embodiments, R₄ is AHB.

According to still further some embodiments R₅ is selected from thegroup consisting of hydroxyl and amine and Y is alkyl.

According to some embodiments, each of the compounds presented hereinhas selective activity towards eukaryotic cells over prokaryotic cells.

According to some embodiments, each of the compounds presented hereinhas no antibacterial activity.

According to yet another aspect of the present invention there isprovided a process of preparing a compound having the general Formula Ias described herein, wherein R₁ in Formula I is a monosaccharide moietyand R₂ and R₃ are each hydroxyl, the process comprising:

(a) coupling a compound having the general Formula III:

wherein:

the dashed line indicates an R configuration or an S configuration;

Y is hydrogen, alkyl or aryl;

each of T₁-T₂ is independently a hydroxyl protecting group;

each of Q₁ and Q₂ is independently an amine protecting group;

Q₃ is selected from the group consisting of an amine protecting groupand an AHB moiety, the AHB moiety comprises at least one of a hydroxylprotecting group and an amine protecting group; and

X is oxygen or sulfur,

with a derivative of a monosaccharide having a leaving group attached atposition 1″ thereof and at least one of a hydroxyl protecting group andan amino protecting group; and

(b) removing each of the hydroxyl protecting groups and the amineprotecting groups, thereby obtaining the compound.

According some embodiments, each of T₁-T₂ is cyclohexanone dimethylketal.

According to yet another aspect of the present invention there isprovided a process of preparing a compound having the general Formula Ias described herein, wherein R₂ in Formula I is a monosaccharide moietyand R₁ and R₃ are each hydroxyl, the process comprising:

(a) coupling a compound having the general Formula IV:

wherein:

the dashed line indicates an R configuration or an S configuration;

Y is hydrogen, alkyl or aryl;

each of T₁-T₄ is independently a hydroxyl protecting group;

each of Q₁ and Q₂ is independently an amine protecting group;

Q₃ is selected from the group consisting of an amine protecting groupand an AHB moiety, the AHB moiety comprises at least one of a hydroxylprotecting group and an amine protecting group; and

X is oxygen or sulfur,

with a derivative of a monosaccharide having a leaving group attached atposition 1″ thereof and at least one of a hydroxyl protecting group andan amino protecting group; and

(b) removing each of the hydroxyl protecting groups and the amineprotecting groups, thereby obtaining the compound.

According to some embodiments, each of T₁-T₄ is O-acetyl.

According to yet another aspect of the present invention there isprovided a process of preparing a compound having the general Formula Ias described herein, wherein R₃ in Formula I is a monosaccharide moietyand R₁ and R₂ are each hydroxyl, the process comprising:

(a) coupling a compound having the general Formula V:

wherein:

the dashed line indicates an R configuration or an S configuration;

Y is hydrogen, alkyl or aryl;

each of T₁-T₂ is independently a hydroxyl protecting group;

each of Q₁ and Q₂ is independently an amine protecting group;

Q₃ is selected from the group consisting of an amine protecting groupand an AHB moiety, the AHB moiety comprises at least one of a hydroxylprotecting group and an amine protecting group; and

X is oxygen or sulfur,

with a derivative of a monosaccharide having a leaving group attached atposition 1″ thereof and at least one of a hydroxyl protecting group andan amino protecting group; and

(b) removing each of the hydroxyl protecting groups and the amineprotecting groups, thereby obtaining the compound.

According to some embodiments, T₁ is 4-methoxy-1-methylbenzene and T₂ isO-benzoyl.

According to some embodiments, the protected monosaccharide has thegeneral Formula VI:

wherein:

the dashed line indicates an R configuration or an S configuration; eachof Z₁, Z₂ and Z₃ is independently selected from the group consisting ofthe hydroxyl protecting group and the amine protecting group; and

L is the leaving group.

According to some embodiments, L in Formula VI is selected from thegroup consisting of p-tolylsulfide (p-thiotoluene), thioethyl andtrichloroacetimidate.

According to some embodiments, each of Z₁-Z₃ in Formula VI is a hydroxylprotecting group.

According to yet another aspect of the present invention there isprovided a process of preparing a compound having the general Formula Ias described herein, wherein R₁ is amine and R₂ and R₃ are eachhydroxyl, the process comprising:

(a) reacting a compound having the general Formula III with triflicanhydride to thereby obtain a trifluoro-methanesulfonate group atposition 3′ thereof;

(b) reacting the compound having the trifluoro-methanesulfonate group atposition 3′ thereof with sodium azide; and

(c) removing each of the hydroxyl protecting groups and the amineprotecting groups, thereby obtaining the compound.

According to yet another aspect of the present invention there isprovided a process of preparing a compound having the general Formula Ias described herein, wherein R₁ is the disaccharide moiety having thegeneral Formula I*, and R₂ and R₃ are each hydroxyl, the processcomprising:

(a) coupling a compound having the general Formula III with a compoundhaving the general Formula III*:

wherein:

the dashed line indicates an R configuration or an S configuration;

Y* is hydrogen, alkyl or aryl;

each of T*1-T*₂ is independently a hydroxyl protecting group;

each of Q*₁ and Q*₂ is independently an amine protecting group;

Q*₃ is selected from the group consisting of an amine protecting groupand an AHB moiety, the AHB moiety comprises at least one of a hydroxylprotecting group and an amine protecting group; and

X* is oxygen or sulfur; and

(b) removing each of the hydroxyl protecting groups and the amineprotecting groups, thereby obtaining the compound.

According to some embodiments, the coupling is effected via a linker,and preferably the linker is an alkyl.

According to some embodiments, each of the amine protecting group isselected from the group consisting of an azido group and a N-phthalimidegroup.

According to some embodiments, the hydroxyl-protecting group is selectedfrom the group consisting of O-acetyl, O-chloroacetyl and O-benzoyl.

According to yet another aspect of the present invention there isprovided a process of preparing a compound having a general Formula I:

wherein:

each of R₁, R₂ and R₃ is independently a monosaccharide moiety, halide,hydroxyl, amine or an oligosaccharide moiety,

X is oxygen or sulfur;

R₄ is (S)-4-amino-2-hydroxybutyryl (AHB);

R₅ is hydroxyl or amine;

Y is hydrogen, alkyl or aryl;

the dashed line indicates an R configuration or an S configuration;

the process is effected by:

reacting a compound having the general Formula I wherein R₄ is hydrogenwith γ-L-Glu-AHB-SNAC in the presence of enzyme BtrH to thereby obtain acompound having the general Formula I wherein R₄ is a γ-L-Glu-AHB; andreacting the compound having the general Formula I wherein R₄ is aγ-L-Glu-AHB with enzyme BtrG to thereby obtain the compound having thegeneral Formula I wherein R₄ is (S)-4-amino-2-hydroxybutyryl (AHB).

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”. The term“consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a biomolecule” or “at least one biomolecule” may include aplurality of biomolecules, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. —Copies of this patent or patent application publication withcolor drawing(s) will be provided by the office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 presents the chemical structures of antibacterial aminoglycosidesdescribed in the background art;

FIG. 2 presents the chemical structures and general synthetic pathwaysfor obtaining exemplary compounds according to the present embodiments,which are based on the skeleton of paromamine and were designed to exertstop-codon mutation read-trough activity and reduced toxicity ascompared to other known aminoglycosides;

FIGS. 3A-D present the results of the in vitro mutation suppression andtranslation comparative assays measured for an exemplary Compound 3 andparomomycin, by expression of a plasmid-based reporter constructcontaining a TGA C nonsense stop mutation between a 25-kDa polypeptideencoding open reading frame (ORF) and a 10-kDa polypeptide encoding ORF,in the presence of the tested compounds and [³⁵S]-methionine, showingthe reaction products separated by SDS-PAGE and quantified using aphosphor-imager for Compound 3 (FIG. 3A) and paromomycin (FIG. 3C), andshowing comparative plots where the mutation suppression values (shownin black dots) and the translation values (shown in white dots),calculated as the relative proportion of the total protein at eachconcentration of the tested compounds out of the total protein expressedin the absence thereof, as measured in triplicates for Compound 3 (FIG.3B) and for paromomycin (FIG. 3D);

FIG. 4 presents the ex-vivo suppression of a nonsense mutation exhibitedby an exemplary compound according to the present embodiments, Compound3, compared with paromomycin and gentamicin, using the p2Luc plasmidcontaining a TGA C nonsense mutation in a polylinker between the renillaluciferase and firefly luciferase genes expressed in COS-7 cells,showing the calculated suppression levels as averages of threeindependent experiments or more for each tested compound at differentconcentrations;

FIGS. 5A-H present the results of the in vitro stop codon suppressionlevels assays, induced by gentamicin (marked by black squares),paromomycin (marked by white squares), Compound 37 (marked by blackcircles) and Compound 3 (marked by white circles) in various nonsensemutation context constructs, p2luc (FIG. 5A), R3381X (Duchenne MuscularDystrophy) (FIG. 5B), R3X (FIG. 5C), R245X (Usher Syndrome) (FIG. 5D),G542X (FIG. 5E), W1282X (Cystic Fibrosis) (FIG. 5F), Q70X (FIG. 5G) andW402X (Hurler Syndrome) (FIG. 5H), and the suppression level wascalculated as the relation between the firefly and the renillaluciferases luminescence of the mutant and of the wild type vectors;

FIG. 6 is a comparative bar-graph, presenting the averages results of atleast three independent experiments of the ex-vivo suppression assays ofR3X nonsense mutation by two exemplary compounds according to thepresent embodiments, Compound 37 and Compound 3, as compared toparomomycin and gentamicin;

FIG. 7 presents the UV melting profiles observed for the humancytoplasmic A site olygonucleotide model duplexes in the absence of anydrug (red curve in FIG. 7) and in complex with ribostamycin (blue curvein FIG. 7), Compound 3 (pink curve in FIG. 7) and Compound 37 (greencurve in FIG. 7) complexes at a drug/duplex ratio of 5:1;

FIGS. 8A-D present a comparison of ¹H NMR spectra (FIGS. 8A and 8B) and¹³C NMR spectra (FIGS. 8C and 8D) of Compound 37 prepared by thechemo-enzymatic procedure presented herein (FIGS. 8A and 8C) and by thechemical procedure presented herein (FIGS. 8B and 8D), whereas the “*”denotes unidentified impurities;

FIGS. 9A-C present a comparison of 2D-COSY spectra of Compound 37prepared by chemical (FIG. 9A) and chemoenzymatic (FIG. 9B) procedurespresented herein, with that of the Compound 3 (FIG. 9C), whereas thedashed lines show correlations between 2-Hax and 2-Heq protons with 1-Hand 3-H protons of the 2-DOS ring, highlighting strong downfield shiftof the 1-H proton in Compound 37 versus 1-H proton in the parentcompound Compound 3.

FIG. 10 is a flow chart depicting the preparation of Compound 2;

FIG. 11 is a flow chart depicting the preparation of Compound 3;

FIG. 12 is a flow chart depicting the preparation of Compound 4;

FIG. 13 is a flow chart depicting the preparation of Compound 5;

FIG. 14 is a flow chart depicting the preparation of Compound 6;

FIG. 15 is a flow chart depicting the preparation of Compound 7;

FIG. 16 is a flow chart depicting the preparation of Compound 8; and

FIG. 17 is a flow chart depicting the preparation of Compound 9.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a new class of aminoglycosides, whichcan be beneficially used in, for example, the treatment of geneticdiseases. Specifically, the present invention is of a new class ofcompounds, derived from paromomycin, which exhibit high prematurestop-codon mutations read-through activity while exerting low toxicityin mammalian cells. The present invention is thus further ofpharmaceutical compositions containing these compounds, and of usesthereof in the treatment of genetic disorders, such as cystic fibrosis.The present invention is further of processes of preparing thesecompounds.

The principles and operation of the present invention may be betterunderstood with reference to the figures and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

As discussed above, over the past decade, many analogs of naturalaminoglycosides have been designed and synthesized to overcome the rapidspread of antibiotic resistance to these drugs in pathogenic bacteria,whereby some of the antibiotic aminoglycosides have been shown to possesstop codon mutation suppression activity. However, nearly all stop codonmutation suppression experiments for the potential use of theseaminoglycosides in the treatment of human genetic diseases have beenperformed with commercially available aminoglycosides, and almost noefforts were made to optimize the activity of these aminoglycosides asstop codon read-through inducers.

To date, there is still no clear answer to the question why someaminoglycosides induce termination suppression, while others do not.Comparison of the in-vitro suppression activity of several commercialaminoglycosides in mammalian system have shown that generallyaminoglycosides with a C6′ hydroxyl group on “Ring I”, such as in G-418and paromomycin (see, FIG. 1) are more effective than those with amineat the same position [6, 51].

While further exploring the effect of aminoglycosides on stop-codonmutation suppression, with the aim of finding aminoglycoside analogsthat would exhibit improved stop-codon read-through activity as well asreduced toxicity, the present inventors have envisioned that separatingelements of the aminoglycoside structure that induce toxicity from thosethat are required for an antibacterial and/or stop-codon mutationsuppression activity would be beneficial to this effect. From theavailable toxicity data on clinically used aminoglycosides and somedesigned structures (see, FIG. 1), it was hypothesized that the two mainfactors that significantly influence the toxicity of aminoglycoside arethe reduction in the number of amino groups (deamination), and/ordeletion of ring hydroxyl groups (deoxygenation).

Reduced toxicity of aminoglycoside as a result of deamination (removalof amino groups) was observes in, for example, paromomycin, whichdiffers from neomycin in that it has one less amino group, and is muchless toxic than neomycin (LD₅₀ of neomycin=24, paromomycin=160). Thus,this difference of one charge (in terms of a positively charged aminegroup at physiological pH) makes a great difference in the toxicity ofthe two compounds. Similarly, one charge difference of kanamycin B(LD₅₀=132) from kanamycin A (LD₅₀=280) and kanamycin C (LD₅₀=225)rendered the latter two less toxic than kanamycin B. Without being boundby any particular theory, it has been assumed that such reduction in thetoxicity of aminoglycosides upon decrease in charged amino groups can beexplained by decrease of nonspecific interaction with other cellcomponents, and by the reduced production of free radicals. Anadditional factor that has been noted to affect the toxicity ofaminoglycosides is acylation of N1-amine of the 2-DOS ring with(S)-4-amino-2-hydroxybutyryl (AHB) group, although the extent of thiseffect has been shown to depend on the aminoglycoside structure (forexample, neamine LD₅₀=125 vs N1-AHB-neamine LD₅₀=260; and kanamycin ALD₅₀=280 vs amikacin LD₅₀=300).

Increased toxicity of aminoglycosides as a result of a deoxygenation(removal of hydroxyl groups) was observed in, for example, the removalof 3′-OH in kanamycin B (LD₅₀=132) to afford tobramycin (LD₅₀=79), whichis much more toxic than the parent kanamycin B. Without being bound byany particular theory, this phenomenon can be explained by reduction inthe basicity of the 2′-NH₂ adjacent to the 3′-OH. Corroborating resultshave been provided by displacement of the 5-OH with 5-fluorine inkanamycin B and its several clinical derivatives [68-70]. Thus,significantly high toxicity of the clinical drugs such as tobramycin(3′-deoxy), gentamicin (3′,4′-dideoxy), dibekacin (3′,4′-dideoxy) andarbekacin (3′,4′-dideoxy) could be ascribed to the increased basicity of2-NH₂ group (“Ring I”) in these drugs caused mainly because the lack ofC3′-oxygen or C3′,C4′-oxygen atoms.

The structural manipulations which were introduced into theaminoglycoside analogs presented in U.S. patent application Ser. No.11/073,649 include, inter alia, the addition of a rigid sugar ring. Theaddition of a rigid sugar ring to the aminoglycoside scaffold affectedthe interaction thereof with resistance-causing enzymes, and thereforecontributed to the inhibition of the formation of a ternary complexrequired for enzymatic catalysis and the subsequent emergence ofresistance.

While reducing the present invention to practice, the present inventorsdesigned and successfully prepared and practiced novel compounds whichexhibit efficient mutation suppression activity and reduced toxicity.These compounds are based on a paromamine scaffold, obtained fromparomomycin by removing two monosaccharide moieties therefrom, to whichnew structural features were introduced. The manipulations of thestructural features of paromomycin were carefully selected in order toreduce potential toxicity and improve mutation read-through activity.

Hence, according to one aspect of the present invention there isprovided a compound having a general Formula I:

wherein:

the dashed line indicates an R configuration or an S configuration;

each of R₁, R₂ and R₃ is independently a monosaccharide moiety, halide,hydroxyl, amine or an oligosaccharide moiety,

X is oxygen or sulfur;

R₄ is hydrogen or an (S)-4-amino-2-hydroxybutyryl (AHB) moiety;

R₅ is hydroxyl or amine; and

Y is hydrogen, alkyl or aryl.

The term “monosaccharide”, as used herein and is well known in the art,refers to a simple form of a sugar that consists of a single saccharidemolecule which cannot be further decomposed by hydrolysis. Most commonexamples of monosaccharides include glucose (dextrose), fructose,galactose, and ribose. Monosaccharides can be classified according tothe number of carbon atoms of the carbohydrate, i.e., triose, having 3carbon atoms such as glyceraldehyde and dihydroxyacetone; tetrose,having 4 carbon atoms such as erythrose, threose and erythrulose;pentose, having 5 carbon atoms such as arabinose, lyxose, ribose,xylose, ribulose and xylulose; hexose, having 6 carbon atoms such asallose, altrose, galactose, glucose, gulose, idose, mannose, talose,fructose, psicose, sorbose and tagatose; heptose, having 7 carbon atomssuch as mannoheptulose, sedoheptulose; octose, having 8 carbon atomssuch as 2-keto-3-deoxy-manno-octonate; nonose, having 9 carbon atomssuch as sialose; and decose, having 10 carbon atoms. Monosaccharides arethe building blocks of oligosaccharides like sucrose (common sugar) andother polysaccharides (such as cellulose and starch).

As used herein, the phrase “moiety” describes a part, and preferably amajor part, of a chemical entity, such as a molecule or a group, whichhas underwent a chemical reaction and is now covalently linked toanother molecular entity.

As used herein, the term “halide” (also referred to herein as “halo”),describes an atom of fluorine, chlorine, bromine or iodine, alsoreferred to herein as fluoride, chloride, bromide and iodide.

The term “hydroxyl”, as used herein, refers to an —OH group.

As used herein, the term “amine” describes a —NR′R″ group where each ofR′ and R″ is independently hydrogen, alkyl, cycloalkyl, heteroalicyclic,aryl or heteroaryl, as these terms are defined herein.

As used herein, the term “alkyl” describes an aliphatic hydrocarbonincluding straight chain and branched chain groups. Preferably, thealkyl group has 1 to 20 carbon atoms, and more preferably 1-10 carbonatoms. Whenever a numerical range; e.g., “1-10”, is stated herein, itimplies that the group, in this case the alkyl group, may contain 1carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including10 carbon atoms. The alkyl can be substituted or unsubstituted. Whensubstituted, the substituent can be, for example, an alkyl, an alkenyl,an alkynyl, a cycloalkyl, an aryl, a heteroaryl, a halide, a hydroxy, analkoxy and a hydroxyalkyl as these terms are defined hereinbelow. Theterm “alkyl”, as used herein, also encompasses saturated or unsaturatedhydrocarbon, hence this term further encompasses alkenyl and alkynyl.

The term “alkenyl” describes an unsaturated alkyl, as defined herein,having at least two carbon atoms and at least one carbon-carbon doublebond. The alkenyl may be substituted or unsubstituted by one or moresubstituents, as described hereinabove.

The term “alkynyl”, as defined herein, is an unsaturated alkyl having atleast two carbon atoms and at least one carbon-carbon triple bond. Thealkynyl may be substituted or unsubstituted by one or more substituents,as described hereinabove.

The term “aryl” describes an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. The aryl groupmay be substituted or unsubstituted by one or more substituents, asdescribed hereinabove.

The term “heteroaryl” describes a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) group having in the ring(s) oneor more atoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. Examples,without limitation, of heteroaryl groups include pyrrole, furane,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline and purine. The heteroaryl group may besubstituted or unsubstituted by one or more substituents, as describedhereinabove. Representative examples are thiadiazole, pyridine, pyrrole,oxazole, indole, purine and the like.

The two-ring structure presented in Formula I above is referred toherein as a paromamine scaffold.

According to some embodiments of the present invention, X is oxygen.

In some embodiments, R₅ is hydroxyl.

In some embodiments, Y is hydrogen or alkyl, and more preferably Y ismethyl.

In one embodiment of the present invention, the paromamine scaffoldpresented in Formula I above comprises one or more additionalmonosaccharide moiety or moieties attached thereto.

As can be seen in Formula I hereinabove, there are several positionsonto which a monosaccharide moiety can be attached to the paromaminescaffold.

According to some embodiments, there are three positions, denoted as R₁,R₂ and R₃, which are preferred for introducing a monosaccharide moietyto the paromamine scaffold.

In one embodiment, at least one of R₁, R₂ and R₃ is a monosaccharidemoiety.

In some embodiments, only one monosaccharide is introduced to any one ofR₁, R₂ or R₃. In such cases, preferably, the other positions arehydroxyls.

In some embodiments, the monosaccharide moiety is introduced at the R₁position, and R₂ and R₃ are preferably each hydroxyl.

Similarly, when a monosaccharide moiety is introduced at the R₂position, R₁ and R₃ are preferably each hydroxyl, and when amonosaccharide moiety is introduced at the R₃ position, R₁ and R₂ arepreferably each hydroxyl.

According to some embodiments, at least one of R₁, R₂ and R₃ is amonosaccharide moiety. Preferably the monosaccharide is a pentose, suchas a furanose, or a hexose, such as a pyranose. Preferred monosaccharidemoieties according to the present embodiments can be collectivelyrepresented by the general Formula II:

wherein each of R₆, R₇ and R₈ is independently selected from the groupconsisting of hydroxyl and amine, and the dashed line indicates an Rconfiguration or an S configuration. The curved line indicates theposition of the monosaccharide moiety that is coupled to the paromaminescaffold.

In some embodiments, R₇ and R₈ are each hydroxyl.

As presented and demonstrated in the Examples section that follows (see,Table 18), the substituent at the R₆ position was found to affect boththe truncation mutation read-though activity and the antimicrobialactivity of the resulting compound. Preferred compounds having amonosaccharide moiety represented by Formula II above therefore have anamine or hydroxyl group at the R₆ position, and more preferably, anamine.

In one embodiment of the present invention, the paromamine scaffoldpresented in Formula I above comprises one or more additionaloligosaccharide moiety or moieties attached thereto, preferably atpositions R₁, R₂ and R₃. Hence, according to preferred embodiments, atleast one of R₁, R₂ and R₃ is an oligosaccharide moiety. Preferably,only one oligosaccharide moiety is introduced to any one of R₁, R₂ orR₃. In such cases, preferably, the other positions are hydroxyls.

Preferably, the oligosaccharide moiety is attached to the R₁ positionwhile the other two positions, R₂ and R₃, are preferably each hydroxyl.Alternatively, the oligosaccharide moiety is coupled to the R₂ position,and preferably, R₁ and R₃ are each hydroxyl, or the oligosaccharidemoiety is coupled to the R₃ position, and preferably, R₁ and R₂ are eachhydroxyl.

The term “oligosaccharide” as used herein refers to a compound thatcomprises two or more monosaccharide units, as these are defined herein.Preferably, the oligosaccharide comprises 2-6 monosaccharides, morepreferably the oligosaccharide comprises 2-4 monosaccharides and mostpreferably the oligosaccharide is a disaccharide moiety, having twomonosaccharide units.

According to some embodiments, the disaccharide coupled to the compoundhaving general Formula I, has general Formula I* as follows:

wherein:

the dashed line indicates an R configuration or an S configuration;

each of R*₁, R*₂ and R*₃ is independently a halide, hydroxyl, amine oris linked to the compound having general Formula I, whereas at least oneof R*₁, R*₂ and R*₃ is linked to the compound having the general FormulaI.

X* is oxygen or sulfur;

R*₄ is hydrogen or an (S)-4-amino-2-hydroxybutyryl (AHB) moiety;

R*₅ is hydroxyl or amine; and

Y* is hydrogen, alkyl or aryl.

Such a “dimer” therefore includes two compounds attached to one anotherat their corresponding R₁, R*₁, R₂, R*₂, R₃ or R*₃ positions in anycombination thereof, for example, an R₁-R*₂ or R2-R*₁ linked dimer, anR₁-R*₃ or R₃—R*₁ linked dimer, an R₃-R*₂ or R₂-R*₃ linked dimer, anR₁-R*₁ linked dimer, an R2-R*₂ linked dimer or an R₃—R*₃ linked dimer.Preferably it is an R₁-R*₁ linked dimer.

The link between the two moieties can be via a linker, or a linkingmoiety. The term “linker”, as used herein refers to a chemical moietywhich is attached to at least two other chemical moieties, hence linkingtherebetween. In the context of the present embodiments the linker ispreferably a low alkyl having 1-6 carbon atoms and more preferably amethylene.

As discussed hereinabove, although increasing the number of amine groupson the paromamine scaffold may have a negative effect in terms oftoxicity, a paromamine analog, having an amine group at the R₁ positionin an inversed stereochemistry as compared to the original displacedhydroxyl group, was prepared as presented in the Examples section thatfollows, in an attempt to investigate the effect of an additional aminein an inverted configuration.

As demonstrated in the Examples section that follows, Compound 8, anexemplary compound according to the present embodiments, having an amineat the R₁ position which replaces a hydroxyl and possesses an invertedconfiguration was prepared and exhibited 6-16 fold lower antimicrobialactivity (see, Table 18 hereinbelow) as compared to paromomycin.

Hence, in another embodiment of the present invention, R₁ is amine.Preferably, R₂ and R₃ are each hydroxyl.

The compounds described hereinabove can be further grouped into severalsubsets, according to the substituents at the R₄, R₅ and Y positions. Asdefined hereinabove, R₄ can be hydrogen or an(S)-4-amino-2-hydroxybutyryl (AHB) moiety, R₅ can be hydroxyl or amine,and Y can be hydrogen or alkyl (preferably methyl) giving together sixpreferred subsets of compounds with respect to R₄ R5 and Y.

Considering R₄, R₅ and Y, in one subset each of R₄ and Y is hydrogen,and R₅ is hydroxyl, giving the compounds shown in FIG. 2. In the othertwo subsets in this respect R₄ is either hydrogen or AHB and there is anamine at the R₅ position and a methyl at the Y position, and in each ofthese compounds the R₅ position can assume either the R or the Sstereo-configuration.

Each of the above subsets can be further divided by its R₄ substituent,being either hydrogen, or an AHB moiety. Alternatively the AHB moietycan be replaced by an α-hydroxy-β-aminopropionyl (AHP) moiety.

In some embodiments of the present invention, the compound has generalFormula I above and a monosaccharide moiety (e.g., a ribofuranose) asone of R₁, R₂ or R₃.

Compounds having general Formula I above, in which Y is hydrogen, R₄ ishydrogen or an AHB moiety and one of R₁-R₃ is a ribofuranose or pyranosemoiety, are also referred to herein as Compounds 3 and 37-41 (see,Scheme 7 hereinbelow); Compounds having general Formula I above, inwhich R₅ is hydroxyl, R₄ is hydrogen or an AHB moiety, one of R₁-R₃ is aribofuranose or pyranose moiety, and Y is methyl, are also referred toherein as Compounds 42-47 (see, Scheme 8 hereinbelow); and Compoundshaving general Formula I above, in which R₅ is amine, R₄ is hydrogen oran AHB moiety, Y is methyl and one of R₁-R₃ is a ribofuranose orpyranose moiety, are also referred to herein as Compounds 48-53 (see,Scheme 8 hereinbelow).

According to some of the present embodiments,(3S,4R,5S,6S)-5-amino-6-((1R,2R,3R,4R)-4,6-diamino-2-((2S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yloxy)-3-hydroxycyclohexyloxy)-2-(hydroxymethyl)-tetrahydro-2H-pyran-3,4-diol(referred to hereinbelow as Compound 2),(3S,4R,5S,6S)-5-amino-6-((1R,2S,3R,4R)-4,6-diamino-3-((2R,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yloxy)-2-hydroxycyclohexyloxy)-2-(hydroxymethyl)-tetrahydro-2H-pyran-3,4-diol(referred to hereinbelow as Compound 4), amikacin, apramycin, arbekacin,butirosin, dibekacin, fortimycin, G-418, gentamicin, hygromycin,habekacin, dibekacin, netlmicin, istamycin, isepamycin, kanamycin,lividomycin, neamine, neomycin, paromomycin, ribostamycin, sisomycin,spectinomycin, streptomycin, tobramycin and any variants thereof havinga suffix added their name, such as, for example, gentamicin C1,gentamicin C1A, gentamicin C2, gentamicin D, kanamycin A, kanamycin B,butirosin A, hygromycin B, neomycin B, etc., have been previouslydescribed and are therefore excluded from the scope of this aspect ofthe present invention. These also include any other aminoglycosideanalogs having two or more monosaccharide units, which have beenpreviously described.

Nonetheless, Compound 2 and Compound 4, were neither described nortested in the context of their therapeutic activity in general, letalone in the context of treatment of genetic disorders, and in thecontext of stop-codon mutation suppression in particular, and aretherefore not excluded from the scope of other aspects of the presentinvention.

The present embodiments further encompass any enantiomers, prodrugs,solvates, hydrates and/or pharmaceutically acceptable salts of thecompounds described herein.

As used herein, the term “enantiomer” refers to a stereoisomer of acompound that is superposable with respect to its counterpart only by acomplete inversion/reflection (mirror image) of each other. Enantiomersare said to have “handedness” since they refer to each other like theright and left hand. Enantiomers have identical chemical and physicalproperties except when present in an environment which by itself hashandedness, such as all living systems.

The term “prodrug” refers to an agent, which is converted into theactive compound (the active parent drug) in vivo. Prodrugs are typicallyuseful for facilitating the administration of the parent drug. They may,for instance, be bioavailable by oral administration whereas the parentdrug is not. A prodrug may also have improved solubility as comparedwith the parent drug in pharmaceutical compositions. Prodrugs are alsooften used to achieve a sustained release of the active compound invivo. An example, without limitation, of a prodrug would be a compoundof the present invention, having one or more carboxylic acid moieties,which is administered as an ester (the “prodrug”). Such a prodrug ishydrolyzed in vivo, to thereby provide the free compound (the parentdrug). The selected ester may affect both the solubility characteristicsand the hydrolysis rate of the prodrug.

The term “solvate” refers to a complex of variable stoichiometry (e.g.,di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by asolute (the compound of the present invention) and a solvent, wherebythe solvent does not interfere with the biological activity of thesolute. Suitable solvents include, for example, ethanol, acetic acid andthe like.

The term “hydrate” refers to a solvate, as defined hereinabove, wherethe solvent is water.

The phrase “pharmaceutically acceptable salt” refers to a chargedspecies of the parent compound and its counter ion, which is typicallyused to modify the solubility characteristics of the parent compoundand/or to reduce any significant irritation to an organism by the parentcompound, while not abrogating the biological activity and properties ofthe administered compound. An example, without limitation, of apharmaceutically acceptable salt would be a carboxylate anion and acation such as, but not limited to, ammonium, sodium, potassium and thelike.

Further according to the present invention, there are provided processesof preparing the compounds described herein.

The synthetic pathways described herein include a reaction between anacceptor and a donor, whereby the term “acceptor” is used herein todescribe the skeletal structure derived from paromamine which has atleast one and preferably selectively selected available (unprotected)hydroxyl group at positions such as C5, C6 and C3′, which is reactiveduring a glycosidation reaction, and can accept a glycosyl, and the term“donor” is used herein to describe the glycosyl.

The term “glycosyl”, as used herein, refers to a chemical group which isobtained by removing the hydroxyl group from the hemiacetal function ofa monosaccharide and, by extension, of a lower oligosaccharide.

The donors and acceptors are designed so as to form the desiredcompounds.

The following describes some embodiments of this aspect of the presentinvention, presenting exemplary processes of preparing exemplary subsetsof the compounds described herein.

The syntheses of the compounds of the present embodiments generallyinclude (i) preparing an acceptor compound by selective protection ofone or more hydroxyls and amines at selected positions present on theparomamine scaffold, leaving one or two positions unprotected andtherefore free to accept a donor (glycosyl) compound as definedhereinabove; (ii) manipulating a structural feature of the acceptor atthe desired position, by e.g., a coupling reaction with a suitablyprotected donor compound to the unprotected position on the acceptor, orby replacing a hydroxyl group with an amine group and optionallyinversing the configuration of the amine; and (iii) removing of allprotecting groups.

The phrase “protecting group”, as used herein, refers to a substituentthat is commonly employed to block or protect a particular functionalitywhile reacting other functional groups on the compound. For example, an“amino-protecting group” is a substituent attached to an amino groupthat blocks or protects the amino functionality in the compound.Suitable amino-protecting groups include azide (azido), N-phthalimido,N-acetyl, N-trifluoroacetyl, N-t-butoxycarbonyl (BOC),N-benzyloxycarbonyl (CBz) and N-9-fluorenylmethylenoxycarbonyl (Fmoc).Similarly, a “hydroxyl-protecting group” refers to a substituent of ahydroxyl group that blocks or protects the hydroxyl functionality.Suitable protecting groups include cyclohexanone dimethyl ketal (forminga 1,3-dioxane with two adjacent hydroxyl groups),4-methoxy-1-methylbenzene (forming a 1,3-dioxane with two adjacenthydroxyl groups), O-acetyl, O-chloroacetyl, O-benzoyl and O-silyl. For ageneral description of protecting groups and their use, see T. W.Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, NewYork, 1991.

According to the embodiments presented hereinbelow, the amine protectinggroups include an azide group and/or an N-phthalimide group, and thehydroxyl-protecting groups include O-acetyl, O-chloroacetyl and/orO-benzoyl.

In one embodiment, there is provided a process of preparing an exemplarysubset of the compounds having the general Formula I as presentedherein, wherein a monosaccharide is attached to the R₁ position and R₂and R₃ are each hydroxyl. The process, according to this embodiment, iseffected by preparing a suitably protected acceptor compound and asuitably protected donor compound, coupling these two compounds to oneanother, and subsequently removing all the protecting groups from theresulting compound.

The acceptor, according to this embodiment, has the general Formula III:

which is a version of paromamine having protecting groups at specificpositions, wherein:

the dashed line indicates an R configuration or an S configuration;

Y is hydrogen, alkyl or aryl;

each of T₁-T₂ is independently a hydroxyl protecting group;

each of Q₁ and Q₂ is independently an amine protecting group;

Q₃ is selected from the group consisting of an amine protecting groupand an AHB moiety, in which the amine and hydroxyl groups are protected;and

X is oxygen or sulfur, preferably oxygen.

The protecting groups are selected such that they are easily attachedand removed under suitable conditions, and according to the differentialreactivity of the various amine and hydroxyl groups of paromamine, suchthat the hydroxyl group at the R₁ position thereof is left unprotectedand free for the coupling reaction.

According to some embodiments, each of T₁-T₂ is a cyclohexanone dimethylketal protecting group, forming a 1,3-dioxane with two adjacent hydroxylgroups in the case of T₁, and forming a 1,3-dioxolane in the case of T₂.

The donor compound is a protected monosaccharide having a leaving groupat position 1″ thereof. Such protected monosaccharides can becollectively represented by the general Formula VI:

wherein each of Z₁, Z₂ and Z₃ is independently selected from the groupconsisting of a hydroxyl protecting group and a amine protecting group,L is the leaving group, and the dashed line indicates an R configurationor an S configuration.

As used herein, the phrase “leaving group” describes a labile atom,group or chemical moiety that readily undergoes detachment from anorganic molecule during a chemical reaction, while the detachment isfacilitated by the relative stability of the leaving atom, group ormoiety thereupon. Typically, any group that is the conjugate base of astrong acid can act as a leaving group. Representative examples ofsuitable leaving groups according to the present embodiments thereforeinclude, without limitation, halide, acetate, tosylate, triflate,sulfonate, azide, hydroxy, thiohydroxy, alkoxy, cyanate, thiocyanate,nitro and cyano.

The term “acetate” refers to acetic acid anion.

The term “tosylate” refers to toluene-4-sulfonic acid anion.

The term “triflate” refers to trifluoro-methanesulfonic acid anion.

The term “azide” refers to an N₃—.

The terms “hydroxy” and “thiohydroxy” refer to the OH⁻ and SH⁻ anionsrespectively.

The term “cyanate” and “thiocyanate” refer to [O═C═N]⁻ and [S═C═N]⁻anions respectively.

The term “nitro” refers to NO₂—.

The term “cyano” refers to [C≡N]—.

Preferably L is p-tolylsulfide (p-thiotoluene), thioethyl andtrichloroacetimidate, and further preferably each of Z₁-Z₃ is a hydroxylprotecting group.

The process is therefore effected by:

(a) coupling the abovementioned acceptor compound to the abovementioneddonor compound; and

(b) subsequently removing each of the protecting groups.

Exemplary compounds which were prepared according to this embodimentinclude Compound 6 and Compound 7, as presented in the Examples sectionthat follows.

This rudimentary process is used to prepare other exemplary subsets ofthe compounds according to the present embodiments, upon utilizing anacceptor that is designed to interact with a donor at a desiredposition.

Hence, according to another embodiment, there is provided a process ofpreparing an exemplary subset of the compounds having the generalFormula I as presented herein, wherein a monosaccharide is attached tothe R₂ position and R₁ and R₃ are each hydroxyl. Such a process iseffected by:

(a) coupling a compound having the general Formula IV:

wherein:

the dashed line indicates an R configuration or an S configuration;

Y is hydrogen, alkyl or aryl;

each of T₁-T₄ is independently a hydroxyl protecting group;

each of Q₁ and Q₂ is independently an amine protecting group;

Q₃ is selected from the group consisting of an amine protecting groupand an AHB moiety, the AHB moiety comprises at least one of a hydroxylprotecting group and an amine protecting group; and

X is oxygen or sulfur,

with a derivative of a monosaccharide having a leaving group attached atposition 1″ thereof and at least one of a hydroxyl protecting group andan amino protecting group; and

(b) removing each of the hydroxyl protecting groups and the amineprotecting groups, to thereby obtain the compound.

Preferably, each of T₁-T₄ is O-acetyl.

As in the previously presented embodiment, the derivative of amonosaccharide is a protected monosaccharide that has the generalFormula VI, as presented hereinabove.

Exemplary compounds which were prepared according to this embodimentinclude Compound 2, Compound 3 and Compound 37, as presented in theExamples section that follows.

According to yet another embodiment there is provided a process ofpreparing an exemplary subset of the compounds having the generalFormula I as presented herein, wherein a monosaccharide is attached tothe R₃ position and R₁ and R₂ are each hydroxyl. Such a process iseffected by:

(a) coupling a compound having the general Formula V:

wherein:

the dashed line indicates an R configuration or an S configuration;

Y is hydrogen, alkyl or aryl;

each of T₁-T₂ is independently a hydroxyl protecting group;

each of Q₁ and Q₂ is independently an amine protecting group;

Q₃ is selected from the group consisting of an amine protecting groupand an AHB moiety, the AHB moiety comprises at least one of a hydroxylprotecting group and an amine protecting group; and

X is oxygen or sulfur,

with a derivative of a monosaccharide having a leaving group attached atposition 1″ thereof and at least one of a hydroxyl protecting group andan amino protecting group; and

(b) removing each of the hydroxyl protecting groups and the amineprotecting groups, to thereby obtain the compound.

Preferably, T₁ is 4-methoxy-1-methylbenzene and T₂ is O-benzoyl.

Exemplary compounds which were prepared according to this embodimentinclude Compound 4 and Compound 5, as presented in the Examples sectionthat follows.

In other embodiments, the acceptors presented hereinabove are utilizedin the preparation of other subsets of compounds having general FormulaI.

Thus, in still another embodiment, there is provided a process ofpreparing a compound having the general Formula I as presentedhereinabove, wherein R₁ is amine which exhibits an inverted stereoconfiguration as compared to the corresponding hydroxyl group in theparent paromamine compound, and R₂ and R₃ are each hydroxyl. Such aprocess is effected by:

(a) reacting a compound having the general Formula III:

wherein:

the dashed line indicates an R configuration or an S configuration;

Y is hydrogen, alkyl or aryl;

each of T₁-T₂ is independently a hydroxyl protecting group;

each of Q₁ and Q₂ is independently an amine protecting group;

Q₃ is selected from the group consisting of an amine protecting groupand an AHB moiety, the AHB moiety comprises at least one of a hydroxylprotecting group and an amine protecting group; and

X is oxygen or sulfur,

with triflic anhydride to thereby obtain a trifluoro-methanesulfonategroup at position 3′ thereof;

(b) reacting the compound having the trifluoro-methanesulfonate group atposition 3′ thereof with sodium azide; and

(c) removing each of the hydroxyl protecting groups and the amineprotecting groups, thereby obtaining the compound having an amine at theR₁ position.

An exemplary compound which was prepared according to this embodimentincludes Compound 8, as presented in the Examples section that follows.

According to an additional embodiment, there is provided a process ofpreparing a dimer compound having the general Formula I as presentedhereinabove, wherein R₁ is the disaccharide moiety having the generalFormula I* described hereinabove, and R₂ and R₃ are each hydroxyl. Sucha process is effected by:

(a) coupling a compound having the general Formula III with anothercompound having the general Formula III*:

wherein:

the dashed line indicates an R configuration or an S configuration;

Y* is hydrogen, alkyl or aryl;

each of T*1-T*₂ is independently a hydroxyl protecting group;

each of Q*₁ and Q*₂ is independently an amine protecting group;

Q*₃ is selected from the group consisting of an amine protecting groupand an AHB moiety, the AHB moiety comprises at least one of a hydroxylprotecting group and an amine protecting group; and

X* is oxygen or sulfur; and

(b) removing each of the hydroxyl protecting groups and the amineprotecting groups, thereby obtaining the dimer compound.

The dimer compound can be a homodimer compound, wherein the twodisaccharides are identical to one another (namely, Y, X, Q₁, Q₂, Q₃, T₁and T₂ and Y*, X*, Q*i, Q*₂, Q*₃, T*₁ and T*₂, respectively, areidentical, or a heterodimer wherein the two disaccharides are differentin one or more features therein.

According to other preferred embodiments of this aspect, the coupling iseffected via a linker, as this term is defined hereinabove. Preferablythe linker is an alkyl, more preferably a low alkyl and most preferablythe linker is a methylene group.

Thus, the coupling is effected in the presence of a bifunctionalcompound, preferably a bifunctional alkyl (e.g., methylene), whichreacts with the two free (unprotected) hydroxyl groups to thereby affectthe coupling therebetween. Such a bifunctional compound preferably hastwo leaving groups, as defined herein.

An exemplary compound which was prepared according to this embodimentincludes Compound 9, as presented in the Examples section that follows.

According to another aspect of the present invention, there is provideda process of preparing a compound having a general Formula I:

wherein:

each of R₁, R₂ and R₃ is independently a monosaccharide moiety, halide,hydroxyl, amine or an oligosaccharide moiety,

X is oxygen or sulfur;

R₄ is (S)-4-amino-2-hydroxybutyryl (AHB);

R₅ is hydroxyl or amine;

Y is hydrogen, alkyl or aryl;

the dashed line indicates an R configuration or an S configuration;

using a chemo-enzymatic reaction.

The process, according to this aspect of the present invention, iseffected by reacting a precursor compound having the general Formula Iwherein R₄ is hydrogen, with γ-L-Glu-AHB-SNAC in the presence of thepurified enzyme BtrH, to thereby obtain an intermediate compound havingthe general Formula I wherein R₄ is a γ-L-Glu-AHB. This intermediatecompound is used, preferably without further purification, in the nextenzymatic reaction with the purified enzyme BtrG, to thereby obtain thecompound having the general Formula I wherein R₄ is(S)-4-amino-2-hydroxybutyryl (AHB).

This process was used as a alternative process for preparing exemplaryCompound 37, as presented in the Examples section that follows.

As demonstrated in the Examples section that follows, the compoundspresented herein were designed so as to, and were indeed shown to,possess a truncation mutation suppression activity, namely the abilityto induce read-through of a stop-codon mutation. Such an activityrenders these compounds suitable for use as therapeutically activeagents for the treatment of genetic disorders, and particularly suchdisorders which are characterized by a truncation mutation.

Thus, according to another aspect of the present invention there isprovided a method of treating a genetic disorder. The method, accordingto this aspect of the present invention, is effected by administering toa subject in need thereof a therapeutically effective amount of one ormore of the compounds presented herein having a general Formula I.

Excluded from the scope of this aspect of the present invention areamikacin, apramycin, arbekacin, butirosin, dibekacin, fortimycin, G-418,gentamicin, hygromycin, habekacin, dibekacin, netlmicin, istamycin,isepamycin, kanamycin, lividomycin, neamine, neomycin, paromomycin,ribostamycin, sisomycin, spectinomycin, streptomycin and tobramycin, andany analogs or variants thereof that have been previously described, asdescribed hereinabove.

As used herein, the terms “treating” and “treatment” include abrogating,substantially inhibiting, slowing or reversing the progression of acondition, substantially ameliorating clinical or aesthetical symptomsof a condition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

As used herein, the phrase “therapeutically effective amount” describesan amount of the polymer being administered which will relieve to someextent one or more of the symptoms of the condition being treated.

The phrase “genetic disorder”, as used herein, refers to a chronicdisorder which is caused by one or more defective genes that are ofteninherited from the parents, and which can occur unexpectedly when twohealthy carriers of a defective recessive gene reproduce, or when thedefective gene is dominant. Genetic disorders can occur in differentinheritance patterns which include the autosomal dominant patternwherein only one mutated copy of the gene is needed for an offspring tobe affected, and the autosomal recessive pattern wherein two copies ofthe gene must be mutated for an offspring to be affected.

According to preferred embodiments the genetic disorder involves a genehaving a truncation mutation which leads to improper translationthereof. The improper translation causes a reduction or abolishment ofsynthesis of an essential protein.

Exemplary such genetic disorders include, but are not limited to, cysticfibrosis (CF), Duchenne muscular dystrophy (DMD), ataxia-telangiectasia,Hurler syndrome, hemophilia A, hemophilia B, Usher syndrome andTay-Sachs.

Accordingly, there is provided a use of a compound having the generalFormula I as presented herein in the manufacture of a medicament fortreating a genetic disorder.

In any of the methods and uses described herein, the compounds describedherein can be utilized either per se or form a part of a pharmaceuticalcomposition, which further comprises a pharmaceutically acceptablecarrier.

Thus, further according to the present invention, there is provided apharmaceutical composition which comprises, as an active ingredient, anyof the novel compounds described herein and a pharmaceuticallyacceptable carrier.

Being primarily directed at treating genetic disorders, which arechronic by definition, the compounds presented herein or pharmaceuticalcompositions containing the same are expected to be administeredthroughout the lifetime of the subject being treated. Therefore, themode of administration of pharmaceutical compositions containing thecompounds should be such that will be easy and comfortable foradministration, preferably by self-administration, and such that willtake the smallest toll on the patient's wellbeing and course of life.

The repetitive and periodic administration of the compounds presentedherein or the pharmaceutical compositions containing the same can beeffected, for example, on a daily basis, i.e. once a day, morepreferably the administration is effected on a weekly basis, i.e. once aweek, more preferably the administration is effected on a monthly basis,i.e. once a month, and most preferably the administration is effectedonce every several months (e.g., every 1.5 months, 2 months, 3 months, 4months, 5 months, or even 6 months).

As discussed hereinabove, some of the limitations for using presentlyknown aminoglycosides as truncation mutation read-through drugs areassociated with the fact that they are primarily antibacterial (used asantibiotic agents). Chronic use of any antibacterial agents is highlyunwarranted and even life threatening as it alters intestinal microbialflora which may cause or worsen other medical conditions such as flaringof inflammatory bowel disease [71], and may cause the emergence ofresistance in some pathological strains of microorganisms [72-75].

The compounds presented herein preferably have no anti-bacterialactivity. By “no anti-bacterial activity” it is meant that the minimalinhibition concentration (MIC) thereof for a particular strain is muchhigher than the concentration of a compound that is considered anantibiotic with respect to this strain. Further, preferably, the MIC ofthese compounds is much higher than the concentration required forexerting truncation mutation suppression activity.

Being preferably non-bactericidal, the compounds presented herein do notsuffer from the aforementioned limitation and hence can be administeredvia absorption paths that may contain benign and/or beneficialmicroorganisms that are not targeted and thus their preservation mayeven be required. This important trait of the compounds presented hereinrenders these compounds particularly effective drugs against chronicconditions since they can be administered repetitively and during lifetime, without causing any adverse, accumulating effects, and can furtherbe administered orally or rectally, i.e. via the GI tract, which is avery helpful and important characteristic for a drug directed attreating chronic disorders.

According to some embodiments, the compounds presented herein areselective towards eukaryotic cells versus prokaryotic cells, namely thecompounds exhibit higher activity in eukaryotic cells, such as those ofmammalian (humans) as compared to their activity in prokaryotic cells,such as those of bacteria. Without being bound by any particular theory,it is assumed that the compounds presented herein, which are known toact by binding to the A-site of the 16S ribosomal RNA while the robosimeis involved in translating a gene, have a higher affinity to theeukaryotic ribosomal A-site, or otherwise are selective towards theeukaryotic A-site, versus the prokaryotic ribosomal A-site.

As used herein a “pharmaceutical composition” refers to a preparation ofthe compounds presented herein, with other chemical components such aspharmaceutically acceptable and suitable carriers and excipients. Thepurpose of a pharmaceutical composition is to facilitate administrationof a compound to an organism.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare: propylene glycol, saline, emulsions and mixtures of organicsolvents with water, as well as solid (e.g., powdered) and gaseouscarriers.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the compounds presentedherein into preparations which, can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen.

According to some embodiments, the most preferred route ofadministration is effected orally. For oral administration, thecompounds presented herein can be formulated readily by combining thecompounds with pharmaceutically acceptable carriers well known in theart. Such carriers enable the compounds presented herein to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, thecompounds presented herein may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for the chosen routeof administration.

For injection, the compounds presented herein may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline bufferwith or without organic solvents such as propylene glycol, polyethyleneglycol.

For transmucosal administration, penetrants are used in the formulation.Such penetrants are generally known in the art.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active aminoglicoside compounds doses.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds presented herein areconveniently delivered in the form of an aerosol spray presentation(which typically includes powdered, liquified and/or gaseous carriers)from a pressurized pack or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compounds presented herein and a suitablepowder base such as, but not limited to, lactose or starch.

The compounds presented herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the compounds preparation in water-soluble form.Additionally, suspensions of the compounds presented herein may beprepared as appropriate oily injection suspensions and emulsions (e.g.,water-in-oil, oil-in-water or water-in-oil in oil emulsions). Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents, which increase the solubility ofthe compounds presented herein to allow for the preparation of highlyconcentrated solutions.

Alternatively, the compounds presented herein may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

The compounds presented herein may also be formulated in rectalcompositions such as suppositories or retention enemas, using, e.g.,conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofcompounds presented herein effective to prevent, alleviate or amelioratesymptoms of the disorder, or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compounds presented herein used in the methods of the presentembodiments, the therapeutically effective amount or dose can beestimated initially from activity assays in animals. For example, a dosecan be formulated in animal models to achieve a circulatingconcentration range that includes the mutation suppression levels asdetermined by activity assays (e.g., the concentration of the testcompounds which achieves a substantial read-through of the truncationmutation). Such information can be used to more accurately determineuseful doses in humans.

Toxicity and therapeutic efficacy of the compounds presented herein canbe determined by standard pharmaceutical procedures in experimentalanimals, e.g., by determining the EC₅₀ (the concentration of a compoundwhere 50% of its maximal effect is observed) and the LD₅₀ (lethal dosecausing death in 50% of the tested animals) for a subject compound. Thedata obtained from these activity assays and animal studies can be usedin formulating a range of dosage for use in human.

The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the compounds presented herein which are sufficient tomaintain the desired effects, termed the minimal effective concentration(MEC). The MEC will vary for each preparation, but can be estimated fromin vitro data; e.g., the concentration of the compounds necessary toachieve 50-90% expression of the whole gene having a truncationmutation, i.e. read-through of the mutation codon. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. HPLC assays or bioassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using the MEC value.Preparations should be administered using a regimen, which maintainsplasma levels above the MEC for 10-90% of the time, preferable between30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the chronic condition tobe treated, dosing can also be a single periodic administration of aslow release composition described hereinabove, with course of periodictreatment lasting from several days to several weeks or until sufficientamelioration is effected during the periodic treatment or substantialdiminution of the disorder state is achieved for the periodic treatment.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA (the U.S. Food and DrugAdministration) approved kit, which may contain one or more unit dosageforms containing the active ingredient. The pack may, for example,comprise metal or plastic foil, such as, but not limited to a blisterpack or a pressurized container (for inhalation). The pack or dispenserdevice may be accompanied by instructions for administration. The packor dispenser may also be accompanied by a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration. Such notice, for example, may be of labelingapproved by the U.S. Food and Drug Administration for prescription drugsor of an approved product insert. Compositions comprising a compoundaccording to the present embodiments, formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition ordiagnosis, as is detailed hereinabove.

Thus, in one embodiment, the pharmaceutical composition is packaged in apackaging material and identified in print, in or on the packagingmaterial, for use in the treatment of a genetic disorder, as definedherein.

In any of the composition, methods and uses described herein, thecompounds can be utilized in combination with other agents useful in thetreatment of the genetic disorder.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Example 1 Chemical Syntheses

Materials and Methods:

¹H NMR, ¹³C NMR, Distortionless Enhancement by Polarisation Transfer (1DDEPT), Total Correlation Spectroscopy (TOCSY), HeteronuclearMultiple-Quantum Correlation (HMQC), and Heteronuclear Multiple-BondCorrelation (HMBC) spectra were recorded on a Bruker Avance™ 500spectrometer. Chemical shifts, reported in ppm, are relative to internalMe₄Si (δ=0.0) with CDCl₃ as the solvent, and to HOD (hydrogen on demand,δ=4.63) with D₂O as the solvent.

Mass spectroscopy analyses were performed on a Bruker Daltonix Apex 3mass spectrometer for Electrospray Ionization Mass Spectrometry (ESIMS)conditions, or on a TSQ-70B mass spectrometer (Finnigan Mat) MALDIMicromass spectrometer under MALDI-TOF conditions usingα-cyano-4-hydroxycinnamic acid matrix.

Reactions were monitored by TLC on Silica gel (Gel 60 F₂₅₄, 0.25 mm,Merck), and spots were visualized by charring with a yellow solutioncontaining (NH₄)₆Mo₇O₂₄.4H₂O (120 grams) and (NH₄)₂Ce(NO₃)₆ (5 grams) in10% H₂SO₄ (800 ml).

Flash column chromatography was performed on Silica gel “Gel 60” (70-230mesh).

All reactions were carried out under an argon atmosphere and usinganhydrous solvents, unless otherwise indicated.

All chemicals, unless otherwise stated, were obtained from commonvendors.

General Synthetic Overview:

A series of new compounds were designed according to the presentembodiments for the treatment of human genetic diseases caused bypremature stop mutations. All compounds were derived from paromaminewhich is derived from paromomycin.

Compounds 2-9 were synthesized following the paths presented in FIG. 2.As can be seen in FIG. 2, the basic syntheses involves direct Lewis acidpromoted cleavage of paromomycin into the disaccharide paromamine,referred to herein as Compound 1, which is then used as a commonstarting material for the preparation of the designed Compounds. Theprotecting groups used in the below syntheses were chosen based on theirease of attachment and removal, and their stability under the reactionconditions. The glycosidation method of thioglycoside usingN-iodosuccinimide (NIS) [76] and the glycosidation method of thetrichloroacetimidate using BF₃ [77] proved to be both rapid andefficient. The benzoate ester protections at the C-2 position of theribofuranoside donors, Compounds 14a, 14b, 15a and 15b, (see, Scheme 1below) were specially designed to allow selective β-glycoside bondformation between “Ring III” and the paromamine moiety throughneighboring group participation in Compounds 2-7.

The reagents and conditions seen in Scheme 1 above include: (a) TfN₃,Et₃N, CuSO₄, in CH₂Cl₂/MeOH/H₂O 3:10:3; (b) Ac₂O (4.2 equivalents),pyridine, −6° C.; (c) cyclohexanone dimethyl ketal, CSA, DMF, 110° C.;(d) BzCl, pyridine; (e) TFA/H₂O 5:3, THF, 40° C.; (f)anisaldehyde-dimethylacetal, CSA, DMF, 50° C.; and the abbreviationsare: Tf=trifluoromethanesulfonyl, CSA=camphor sulfonic acid,DMF=dimethylformamide, Bz=benzoyl, TFA=trifluoroacetic acid,PMP=p-methoxyphenyl.

Preparation of Compounds 2-7 employed the appropriately protected threedifferent paromamine acceptors, i.e. Compounds 11-13, which selectivelyexpose the hydroxyl groups of the paromamine moiety at positions C5, C6and C3′, to glycosidation reactions, making C5 hydroxyl most susceptiblefor reaction and C3′ hydroxyl least susceptible. These acceptormolecules were readily accessible from the paromamine (Compound 1) asillustrated in Scheme 1 above.

Regioselective acetylation of Compound 10 with acetic anhydride at lowtemperature gave the acceptor Compound 11 at 65% yield [78]. In anotherpathway, treatment of Compound 10 with cyclohexanone dimethyl ketal gavethe second acceptor, Compound 12, in which all functional groups, exceptthe hydroxyl group at position C3′, were protected. Benzoylation ofCompound 12 was followed by acid hydrolysis and benzylidene acetalformation steps to afford the third acceptor Compound 13 at isolatedyield of 86% for all three steps.

The paromamine acceptors Compounds 11-13 were then separately subjectedto glycosidation reactions with two sets of glycosyl donors, i.e.,Compounds 14a-14b and Compounds 15a-b, to furnish the designed protectedderivatives Compounds 16-18 at an overall yield of 68-95%, asillustrated in Scheme 2 below. As presented hereinbelow, the structuresof Compounds 16-18 were confirmed by combination of variousspectroscopic techniques, including HMQC, HMBC, 2D-COSY, and 1D TOCSYNMR spectroscopy. These protected compounds were then subjected toeither two-steps or three-steps deprotection: removal of all the estergroups by treatment with methylamine (33% solution in EtOH), reductionof all the azide groups by the Staudinger reaction, and hydrolysis ofO-benzylidine acetal and cyclohexylidene ketal with aqueoustrifluoroacetic acid, to furnish the final Compounds 2-7, as seen inScheme 2 below, with excellent purity and isolated yields.

The reagents and conditions seen in Scheme 2 above include: (a) Compound15a or Compound 15b, BF₃-Et₂O (catalytic amount), CH₂Cl₂, 4 Å molecularsieves, Compound 11→Compound 16a (85%), Compound 11→Compound 16b (71%),Compound 12→Compound 18a (95%), Compound 12→Compound 18b (93%); (b) (i)MeNH₂ (33% solution in EtOH), (ii) PMe₃(1 M in THF), NaOH 0.1 M, THF,room temperature; Compound 16a→Compound 2 (84%), Compound 16b→Compound 3(91%), Compound 17a→Compound 4 (for 2 steps 75%), Compound 17b→Compound5 (44%), Compound 18a→Compound 6 (84%), Compound 18b→Compound 7 (75%);(c) Compound 14a or Compound 14b NIS, TfOH (catalytic amount), CH₂Cl₂, 4Å molecular sieves, Compound 13→Compound 17a (68%), Compound 13→Compound17b (76%); (d) AcOH/H₂O 6:1, THF 50° C. for Compound 17a, TFA/H₂O 3:2,THF, 60° C. for Compound 17b (52%), AcOH/H₂O 10:3, 1,4-dioxane, 70° C.for Compound 18a (75%), TFA/H₂O 5:1, THF, 50° C. for Compound 18b (82%);and the abbreviations are: PMP=p-methoxyphenyl, NIS=N-iodosuccinimide,Tf=trifluoromethanesulfonyl, CSA=camphor sulfonic acid,DMF=dimethylformamide, Bz=benzoyl, TFA=trifluoroacetic acid.

Scheme 3 below illustrates the preparation of Compounds 8-9. Ring I inCompound 8 having D-allo configuration was prepared from Compound 1 byselectively inverting the configuration at the C3′ position. Triflationof the hydroxyl group at the C3′ position in Compound 12 was followed bynucleophilic displacement with azide to afford the correspondingcis-diazide Compound 19 at a yield of 82% for the two steps. Hydrolysisof the cyclohexylidene ketals with aqueous acetic acid, followed by atwo-step deprotection as described above provided the designed Compound8 at 68% yield. Treatment of the same acceptor Compound 12 with CH₂Br₂in the presence of NaH gave the protected dimmer Compound 20 at a yieldof 82%, which after the similar three-step deprotection as in the caseof Compound 19 afforded the desired dimmer Compound 9 at 86% yield.

The reagents and conditions seen in Scheme 2 above include: (a) Tf₂O,pyridine, (92%); (b) NaN₃, DMF, HMPA, (72%) (c) for Compound 19 AcOH/H₂O8:1,1,4-dioxane, 75° C., (60%), for Compound 20 TFA/H₂O 5:6, THF, 60°C., (90%); (d) (i) MeNH₂ (33% solution in EtOH), (ii) PMe₃ (1 M in THF),NaOH 0.1 M, THF, room temperature; Compound 19→Compound 8 (76%),Compound 20→Compound 9 (81%), (e) CH₂Br₂, NaH, DMF/HMPA 2:1, 4 Åmolecular sieves, (82%), and the abbreviations are:HMPA=hexamethylphosphoramide.

Being based on Compound 10, intermediate Compound 10-AHB (see, Scheme 4below) has the paromamine core but in addition it contains(S)-4-amino-2-hydroxybutyryl (AHB) substitution at the N1 position. TheN1-AHB substitution is expected to further improve both the read-throughactivity and toxicity. This expectation is supported by the recentobservation that amikacin functions better than gentamicin forrestoration of the CFTR protein [79]. Kanamycin A, which differs fromamikacin by only the absence of AHB substitution at N-1 position, doesnot show any read-through activity [6].

Intermediate Compounds 21 and 23 (see, Scheme 4 below) correspond toG-418 and gentamicin respectively. Compounds 21 and 23 are used toprepare C6′-diastereomeric-mixed and separated C6′-diastereomers so asto achieve better nephrotoxicity and cytotoxicity. While thisstereochemical issue was tested on the gentamicin C₂ [68], noC6′-diastereomer of G-418 appears in the literature. 3′-OH and 4′-OHgroups are added to Compound 23 in an attempt to further reduce thetoxicity as observed for gentamicin C₂. In attempts to further modify“Ring II” in Compounds 21 and 23 for structure-activity studies,intermediate Compounds 22 and 24, which combine the functional groups ofeither G-418 and amikacin (corresponding to Compound 22) or gentamicinand amikacin (corresponding to Compound 24) in one molecule.

The preparation of all five Compounds 10-AHB, 21, 22, 23 and 24 startswith paromamine (Compound 1) as a common starting material, readilyaccessible from paromomycin [80]: selective protection of Compound 1 atN2′ and N3 with Cbz is followed by treatment with activated ester of AHB(NOS-AHB-Cbz) according to published procedures [81-84], so as to affordthe corresponding N1-AHB derivative of Compound 1, and treatment of thisintermediate with Pd/H₂ affords Compound 10-AHB.

Similar steps for the introduction of AHB to paromamine 1 followed byselective acetylation gives C5-acceptor Compound 25, and Coupling ofCompound 25 with the trichloroacetamidate donor of 5-azidoribose [80]followed by deprotection steps, afford the designed pseudo-trisaccharideCompound 37 (also referred to herein interchangeably as NB54), asillustrates Scheme 5 below. Similar steps for the introduction of AHB toG-418 produce the intermediate Compound 26 which, after subsequentdeprotection steps, afford its N1-AHB analog Compound 27 (see, Scheme5).

The reagents and conditions seen in Scheme 5 above include: (a) (i)Cu(OAc)₂, Ni(OAc)₂, Cbz-NOS; (ii) NOS-AHB-Cbz, DCC, HOBT; (iii) 4.2equiv. Ac₂O, Py, −7° C.; (b) (i) BF₃—OEt₂, DCM/MeCN; (ii) MeNH₂ (33% solin EtOH); (iii) Pd/C, H₂, dioxane, AcOH; (c) (i) Zn(OAc)₂, Cbz-NOS; (ii)NOS-AHB-Cbz, DCC, HOBT; and (d) Pd/C, H₂, dioxane, AcOH.

For the preparation of Compound 21, paromamine is subjected to asequence of seven steps to afford Compound 28 as a mixture ofC6′-diastereomers, as illustrated in Scheme 6 below. To determine theabsolute stereochemistry of these diastereomers, each is treated withbenzaldehyde dimethyl acetal to afford the corresponding benzylideneacetals, Compound 29 with either an equatorial or an axial C6′-methylgroup. The NOE spectra of these methyl groups with the C5′-proton alongwith the coupling constant of the C6′-proton in each diastereomer allowthe determination of the absolute configuration at C6′-center.Similarly, the absolute configuration at C6′ of the gentamicinderivative Compound 23 is determined. To this effect, Compound 28 isfirst converted to the corresponding amine Compound 30, which, afterprotection with Troc and treatment with NaH, affords the correspondingcyclic oxazolidinone Compound 31. Each diastereomer of Compound 28 andCompound 30 is thereafter subjected to Staudinger reaction followed by asimilar set of reactions as is shown in Scheme 5 hereinabove, to affordthe corresponding N1-AHB derivatives Compound 22 and Compound 24.

The reagents and conditions seen in Scheme 6 above include: (a) (i)TfN₃, Cu(II); (ii) TIPSCl, Py; (iii) PMBCl, NaH, DMF; (iv) HF/Py; (v)Swern oxydation; (vi) MeMgBr, Et₂O; (vii) TFA; (b) Ph(OMe)₂, CSA; (c)(i) cyclohexanone dimethyl ketal, CSA; (ii) Swern oxydation; (iii) NH3,NaBCNH₃, MeOH; (iv) AcOH, MeOH/H₂O; and (d) (i) TrocCl, DCM, Et₃N; (ii)NaH, DMF.

Using similar synthetic routes, three series of compounds, presented inScheme 7 and Scheme 8 below, are prepared using corresponding acceptorcompounds for each series of Compounds, namely the intermediate Compound32 (Scheme 7) for the first series, intermediate Compound 33 for thesecond series and intermediate Compound 34 for the third series (Scheme8) and three different donor Compounds 14b, 35 and 36. Donor Compounds35 and 36 are especially designed with ether protections at C2-OHposition (p-methoxy benzyl in Compound 35 and benzyl in Compound 36) inorder to allow the desired 1,2-cis glycosidic linkage formation.

Donor Compound 35 was obtained in a good isolated yield from gentamicinin the following steps: MeOH, AcCl, reflux (90% yield); TrocCl, NaHCO₃,CHCl₃/H₂O (93% yield); BzCl, Py (70% yield); TolSH, BF₃-Et₂O (53%yield); MeNH₂ in EtOH (90% yield); NaH, PMBCl, TBAI (82% yield). Theoxazolidinone protection in Compound 35 proved to be very efficientunder standard glycosylation reactions. This oxazolidinone ringundergoes spontaneous opening under basic (NaOH) Staudinger conditionwith heating. Thus, as it generally illustrated in Scheme 7 above,coupling of the acceptor 32 with either of the donors, 14b, 35 or 36followed by the deprotection steps afforded the desired Compounds 3,37-41.

Donor Compound 35 was obtained in a good isolated yield from gentamicinin the following steps: MeOH, AcCl, reflux (90% yield); TrocCl, NaHCO₃,CHCl₃/H₂O (93% yield); BzCl, Py (70% yield); TolSH, BF₃-Et₂O (53%yield); MeNH₂ in EtOH (90% yield); NaH, PMBCl, TBAI (82% yield). Theoxazolidinone protection in Compound 35 proved to be very efficientunder standard glycosylation reactions. This oxazolidinone ringundergoes spontaneous opening under basic (NaOH) Staudinger conditionwith heating. Thus, as illustrated in Scheme 7 above, coupling of theacceptor Compound 32 with either of the donors, i.e., Compounds 14b, 35or 36 followed by deprotection steps affords the desired Compounds 3,37-41.

Similarly, coupling of these donors, Compounds 14b, 35 and 36, withappropriate acceptors, i.e., Compounds 33 or 34 (NIS, AgOTf,Et₂O/CH₂Cl₂) provides the corresponding protected compounds which afterstandard deprotection steps affords the desired C5- and C6-linkedderivatives of Compounds 42-53 as illustrated in Scheme 8 below.

The following examples present detailed synthetic procedures forpreparing compounds leading to Compounds 2-9, as outlined in Schemes 1-3above.

Preparation of Paromamine (Compound 1)

Compound 1 was prepared by direct Lewis acid promoted cleavage ofparomomycin according to the published procedure of Ding and co-workers[78] with some modifications.

Acetyl chloride (35 ml) was added to a stirred solution of anhydrousmethanol (215 ml) over 10 minutes at 0° C. After stirring for about 15additional minutes, a commercially available paromomycin sulfate sample(25 grams, 31.0 mmol) was added and the reaction was heated to 70° C.under reflux. Propagation of the reaction was monitored by TLC, using amixture of CH₂Cl₂/MeOH/H₂O/MeNH₂ at a relative ratio of 10:15:6:15diluted to 33% solution in ethanol as eluent, which indicated completionafter 4 hours. The reaction mixture was cooled for about 2 hours in afreezer, filtered, and the residue was dissolved in a minimal amount ofwater. This concentrated aqueous solution was added dropwise to coldethanol (500 ml, 0° C.) and the resulting emulsion was placed in afreezer for about 2 hours. The mixture was filtered and the residue wasdried under vacuum to yield Compound 1 as a white solid (12.5 grams, 94%yield).

¹H NMR (500 MHz, D₂O, pH=3.5) data of Compound 1 are summarized in theTable 1 below.

TABLE 1 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.54 d 3.28 dd 3.80 t 3.70-3.74m 3.35 t 3.78-3.82 m 3.62 dd J = 4.0 J = 4.0, 11.0 J = 10.0 J = 9.0 J =4.0, 12.0 H1 H2eq H2ax H3 H4 H5 H6 II 3.16-3.22 m 2.51 dt 1.72 ddd3.33-3.39 m 3.47 t 3.54 t 3.72 t J = 4.5, 12.5 J₁ = J₂ = J = 9.5 J = 9.0J = 9.5 J₃ = 12.5

¹³C (NMR 125 MHz, D₂O): δ==□□30.6 (C-2), 50.6, 51.6, 55.9, 62.1 (C-6′),71.0, 71.2, 74.3, 75.2, 76.6, 82.7, 98.8 (C-1′);

MALDI TOFMS calculated for C₁₂H₂₅N₃O₇ Na ([M+Na]⁺) m/e: 347.2; measuredm/e: 347.2.

Preparation of Compound 10

Compound 10 was prepared from Compound 1 (paromamine) according to apublished procedure [85] which effected simultaneous conversion of allthe amine groups of Compound 1 into corresponding azide groups bytreatment with TfN₃ to afford Compound 10 at a 90% yield.

Preparation of Compound 11

Compound 11 was prepared from the perazido derivative, Compound 10, byregioselective acetylation with acetic anhydride at low temperatureaccording to the published procedure of Swayze and co-workers [78] withthe following modifications.

Compound 10 (2 grams, 5 mmol), prepared according to a publishedprocedure [85], was dissolved in dry pyridine (5 ml) and the resultedmixture was cooled down to −6° C. and acetic anhydride (4.2 equivalents,2.65 ml) was thereafter added thereto. Propagation of the reaction wasmonitored by TLC (EtOAc/Hexane, 2:3), which indicated completion after 8hours. The reaction was diluted with EtOAc and extracted with HCl (2%),saturated aqueous NaHCO₃, and brine. The combined organic layer wasdried over MgSO₄ and concentrated under reduced pressure. The crudeproduct was purified by flash chromatography (silica gel, EtOAc/Hexane)to yield Compound 11 (1.84 grams, 65% yield).

MALDI TOFMS calculated for C₂₀H₂₇N₉O₁₁ Na ([M+Na]⁺) m/e: 592.2; measuredm/e: 592.2.

Preparation of Compound 12

Cyclohexanone dimethyl ketal (30 ml, 200 mmol) and a catalytic amount ofcamphor sulfonic acid (CSA) were added to a solution of Compound 10 (8.9grams, 22.2 mmol) in dry DMF (30 ml). The reaction was stirred for 1hour at 50° C. and propagation was monitored by TLC (100% EtOAc), whichindicated complete consumption of the starting material. Thereafter thereaction was heated to 110° C. in an oil bath and the propagation of thereaction was monitored by TLC (EtOAc/Hexane, 2:3), which indicated thecompletion of the reaction after 4 hours. The reaction mixture wasdiluted with EtOAc and extracted with saturated aqueous NaHCO₃, brine,dried over MgSO₄ and concentrated under reduced pressure. The crudeproduct was purified by flash chromatography to yield Compound 12 (8.5grams, 67% yield).

¹H NMR (500 MHz, CDCl₃): δ=1.25-1.95 (m, 20H, cyclohexanones), 1.47(ddd, 1H, J₁=J₂=J₃=12.5 Hz, H-2 axial), 2.33 (dt, 1H, J₁=5, J₂=14.5H-2equatorial), 3.26 (dd, 1H, J₁=3.5 J₂=13.5 Hz, H-2′), 3.39 (t, J=9.5 Hz,1H, H-4), 3.47-3.55 (m, 2H, H-3, H-5), 3.55 (t, J=9.5 Hz, 1H, H-5′),3.62-3.68 (m, 1H, H-1), 3.75-3.82 (m, 1H, H-6′), 3.80 (t, J=9.0 Hz 1H,H-6), 3.85-3.92 (m, 2H, H-4′, H-6′), 4.07 (t, 1H, J=9.5 Hz, H-3′), 5.50(d, 1H, J=4.0 Hz, H-1′).

¹³C NMR (125 MHz, CDCl₃): δ=(the range 22.4-37.8 relates tocyclohexanone carbon atoms if not indicated otherwise) 22.5, 22.8, 23.7(2C), 24.9, 25.5, 27.8, 33.9, 36.0, 36.3, 37.8, 57.2, 60.4, 61.5 (C-6′),61.9, 64.0, 68.7, 73.7, 76.8, 79.3, 79.4, 97.0 (C-1′), 99.1 (OCOcyclohexanone ketal), 113.7 (OCO cyclohexanone ketal).

ESIMS calculated for C₂₄H₃₅N₉O₇Na ([M+Na]⁺) m/e: 584.3; measured m/e:584.3.

Preparation of Compound 13e

Compound 12 (2.5 grams, 4.45 mmol) was dissolved in dry pyridine (20 ml)followed by the addition of 4-dimethylamonpyridine (0.5 gram, 4.6 mmol).The reaction mixture was stirred for 5 minutes at room temperature, andthereafter benzoylchloride (1.3 ml, 0.9 mmol) was added. Propagation ofthe reaction was monitored by TLC (EtOAc/Hexane, 1:4), which indicatedcompletion after about 8 hours. The reaction mixture was diluted withEtOAc, extracted with HCl (2%), H₂O and brine, dried over MgSO₄ andconcentrated under reduced pressure.

The crude residue was dissolved in THF (20 ml) added with TFA (5 ml) andwater (3 ml). The reaction mixture was stirred at 40° C. for 8 hoursduring which the propagation of the reaction was monitored by TLC(EtOAc/Hexane, 7:3). The reaction mixture was purified directly by flashchromatography (silica, EtOAc/Hexane) without any further work up toyield Compound 13e (2 grams, 89% overall yield).

¹H NMR (500 MHz, CDCl₃): δ=□□1.49-1.53 (m, 1H, H-2 axial), 2.32-2.35 (m,1H, H-2 equatorial), 3.34-3.44 (m, 4H, H-1, H-3, H-4 and H-5), 3.55-3.57(m, 1H, H-6), 3.74-3.77 (m, 1H, H-2′), 3.86-3.93 (m, 3H, H-5′ and2H-6′), 4.10-4.14 (m, 1H, H-4′), 5.49 (d, 1H, J=3 Hz, H-1′) 5.59 (t, 1H,J=10 Hz, H-3′), 7.48 (t, 2H, J=7.5 Hz), 7.62 (t, 1H, J=7 Hz), 8.08 (d,2H, J=7.5 Hz).

¹³C NMR (125 MHz, CDCl₃): δ=32.0 (C-2), 58.8, 59.6, 61.7 (C-6′), 62.3,69.6, 72.6, 75.1, 75.5, 75.9, 83.2, 99.0 (C-1′), 128.6 (2C), 130.0 (2C),133.9, 167.3.

MALDI TOFMS calculated for C₁₉H₂₃N₉O₈ Na ([M+Na]⁺) m/e: 528.2; measuredm/e: 528.2.

Preparation of Compound 13

1-(Dimethoxymethyl)-4-methoxybenzene (1.3 ml, 7.6 mmol) and a catalyticamount of CSA were added to a solution of Compound 13e (1.93 grams, 3.82mmol) dissolved in dry DMF (10 ml). The reaction mixture was stirred at50° C. and propagation was monitored by TLC (EtOAc/Hexane, 1:1), whichindicated the completion after 8 hours. The reaction was diluted withEtOAc and extracted with saturated NaHCO₃, brine, dried over MgSO₄ andconcentrated under reduced pressure. The crude product was purified byflash chromatography to yield Compound 13 (2 grams, 84% yield).

¹H NMR (500 MHz, CDCl₃): δ=1.47-1.53 (m, 1H, H-2 axial), 2.32-2.35 (m,1H, H-2 equatorial), 3.33-3.45 (m, 4H, H-1, H-3, H-4 and H-5), 3.58-3.61(m, 1H, H-6), 3.74-3.77 (m, 1H, H-2′), 3.75-3.81 (m, 2H, H-5′ and H-6′),3.76 (s, 3H, OCH₃), 4.30-4.35 (m, 2H, H-4′ and H-6′), 5.36 (d, 1H, J=3.5Hz, H-1′), 5.48 (s, 1H), 5.88 (t, 1H, J=10 Hz, H-3′), 6.83 (d, 2H, J=8.5Hz), 7.33 (d, 2H, J=9 Hz), 7.46 (t, 2H, J=7.5 Hz), 7.59 (t, 1H, J=7.5Hz), 8.07 (d, 2H, J=7.5 Hz).

¹³C NMR (125 MHz, CDCl₃): δ=31.9 (C-2), 55.2, 58.5, 59.7, 62.9 (C-5′),63.6 (C-4′), 68.6 (C-6′), 70.5 (C-3′), 75.5, 75.8, 79.2, 83.2, 99.9(C-1′), 101.5, 127.4 (4C), 128.4 (2C), 129.2 (2C), 129.9 (2C), 133.4,160.0, 165.5.

MALDI TOFMS calculated for C₂₇H₂₉N₉O₉ Na ([M+Na]⁺) m/e: 623.2; measuredm/e: 623.2.

Preparation of p-Methylphenyl-2,3,5-tri-O-benzoyl-1-thio-D-ribofuranose(Compound 14a)

4-Methylbenzenethiol (0.6 grams, 4.83 mmol) and BF₃-Et₂O (1.5 ml) wereadded to a solution of 1-O-Acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose(2.0 grams, 3.96 mmol) dissolved in CH₂Cl₂ (25 ml). The resultingmixture was stirred at room temperature under argon. Propagation of thereaction was monitored by TLC (EtOAc/Hexane, 1:4), which indicatedcompletion after 8 hours. The reaction mixture was diluted with EtOAc(200 ml), neutralized with saturated NaHCO₃, and washed with brine. Thecombined organic layer was dried over MgSO₄ and evaporated under reducedpressure. The residue was purified by flash chromatography (silica gel,EtOAc/Hexane) to yield Compound 14a (2.0 grams, 89% yield) as a mixtureof anomers (α/β 3:5).

Spectral Analysis of the α-Anomer:

¹H NMR (500 MHz, CDCl₃) □: δ=2.23 (s, 3H, Me-STol), 4.50 (dd, 1H,J₁=3.5, J₂=9.0 Hz, H-5′), 4.64 (m, 2H, H-4 and H-5), 5.56 (d, 1H, J=5.0Hz, H-1), 5.66 (t, 1H, J=5.0 Hz, H-2), 5.73 (t, 1H, J=5.0 Hz, H-3),7.06-8.11 (19H);

¹³C NMR (125 MHz, CDCl₃): δ=21.1 (Me-STol), 64.3 (C-5), 72.4 (C-3),74.3C-2), 80.4 (C-4), 88.0 (C-1), 128.4-166.2 (27C).

Spectral Analysis of the β-Anomer:

¹H NMR (500 MHz, CDCl₃): δ=2.33 (s, 3H, Me-STol), 4.63 (dd, 1H, J=3.5,J₂=14.0 Hz, H-5), 4.74 (dd, 1H, J₁=3.0 J₂=12.0 Hz, H-5′), 4.88 (dd, 1H,J₁=4.5, J₂=8.0 Hz, H-4), 5.80-5.83 (m, 2H, H-2, H-3), 6.05 (d, 1H, J=5.0Hz, H-1), 7.06-8.11 (19H);

¹³C NMR (125 MHz, CDCl₃): δ=21.1 (Me-STol), 63.8 (C-5), 71.6 (C-3), 72.2(C-2), 79.0 (C-4), 90.8 (C-1), 128.4-166.2 (27C).

MALDI TOFMS calculated for C₃₃H₂₈O₇S Na ([M+Na]⁺) m/e: 591.2; measuredm/e: 591.3.

Preparation of5-deoxy-5-azido-2,3-di-O-benzoyl-1-O-tricloroacetymido-D-ribofuranose(Compound 15b)

N-Bromosuccinimide (NBS, 0.8 grams, 4.41 mmol) was added to a solutionof Compound 14b (1.8 grams, 3.67 mmol), prepared according to apublished procedure [86], in acetone (30 ml) cooled to −10° C., and thepropagation of the reaction was monitored by TLC (EtOAc/Hexane, 3:7),which indicated completion after 2 hours. The reaction mixture wasdiluted with EtOAc (200 ml) and washed with NH₄Cl and brine. Thecombined organic layer was dried over MgSO₄ and evaporated under reducedpressure. The residue was purified by flash chromatography (silica gel,EtOAc/Hexane) to afford the desired anomeric alcohol.

The intermediate alcohol was dissolved in dry CH₂Cl₂ (10 ml) and CCl₃CN(1.7 ml, 11.8 mmol) and K₂CO₃ (200 mg, 1.4 mmol) were added thereto. Themixture was stirred at room temperature and the propagation of thereaction was monitored by TLC (EtOAc/Hexane, 3:7), which indicatedcompletion after 8 hours. The reaction mixture was diluted with CH₂Cl₂,and filtered through celite. The celite was washed thoroughly withCH₂Cl₂, and evaporated to dryness to yield Compound 15b (1.89 grams, 97%overall yield) as a mixture of anomers (α/β 1:9).

Spectral Analysis of the α-Anomer:

¹H NMR (500 MHz, CDCl₃): δ=3.63 (dd, 1H, J₁=5.0, J₂=13.5 Hz, H-5), 3.73(dd, 1H, 1H, J₁=3.5, J₂=13.5 Hz, H-5′), 4.64-4.68 (m, 1H, H-4), 5.76(dd, 1H, J₁=5.0, J₂=6.5 Hz, H-3), 5.94 (d, 1H, J=5.0 Hz, H-2), 6.57 (s,1H, H-1), 7.31-8.15 (10H), 8.72 (s, 1H);

¹³C NMR (125 MHz, CDCl₃): δ=52.9 (C-5), 71.9 (C-3), 74.8C-2), 81.9(C-4), 102.6 (C-1), 128.4-133.6 (10C), 160.4, 165.0, 165.4.

ESIMS calculated for C₁₉H₁₇N₃O₆ Na ([M-C₂NCl₃+Na]⁺) m/e: 406.1; measuredm/e: 406.1.

Preparation of Compound 16a

Anhydrous CH₂Cl₂ (5 ml) was added to powdered, flame-dried 4 Å molecularsieves (500 mg), followed by the addition of the acceptor Compound 11(300 mg, 0.527 mmol), prepared as presented hereinabove, and the donorCompound 15a (1.15 grams, 1.896 mmol), prepared according to a publishedprocedure [86]. The reaction mixture was stirred for 10 minutes at roomtemperature, and then cooled to −40° C. Thereafter a catalytic amount ofBF₃-Et₂O (10 μl) was added to the reaction mixture and stirringcontinues at −15° C. Propagation of the reaction was monitored by TLC(EtOAc/Hexane, 3:7), which indicated completion after 1.5 hours. Thereaction mixture was diluted with CH₂Cl₂, and filtered through celite.After thorough washing of the celite with CH₂Cl₂, the washes werecombined and extracted with saturated aqueous NaHCO₃, brine, dried overMgSO₄ and concentrated. The crude product was purified by flashchromatography to yield Compound 16a (452 mg, yield of 85%).

¹H NMR (500 MHz, CDCl₃) data of Compound 16a are summarized in Table 2below.

TABLE 2 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.69 d 3.44 dd 5.33 t 5.01 t4.42-4.45 m 4.12 dd 4.20 dd J = 4.0 J = 4.0, 10.5 J = 9.5, 10.5 J = 10.0J = 4.5, 12.0 J = 4.5, 12.5 III 5.56 S 5.63 d 5.71-5.74 m 4.67-4.70 m4.36 dd 5.04 dd J = 4.5 J = 3.5, 12.0 J = 3.0, 12.0 H1 H2eq H2ax H3 H4H5 H6 II 3.32-3.39 m 2.31 dt 1.37 ddd 3.32-3.39 m 3.16 t 3.74 t 4.74 t J= 5.0, 13.0 J₁ = J₂ = 12.0 J = 9.0 J = 10.0 J = 10.0 J₃ = 13.0

Additional ¹H NMR (500 MHz, CDCl₃) data for Compound 16a included:δ=2.03 (s, 3H, Ac), 2.06 (s, 3H, Ac), 2.12 (s, 3H, Ac), 2.33 (s, 3H,Ac), 7.35 (t, 2H, J=8, 7.5 Hz, Bz), 7.41 (t, 2H, J=7.5, 8 Hz, Bz),7.49-7.62 (m, 5H, Bz), 7.89 (d, 2H, J=7.5, Bz), 7.95 (d, 2H, J=7, Bz),8.14 (d, 2H, J=7, Bz).

¹³C NMR (125 MHz, CDCl₃): δ=20.6, 20.7, 20.8, 21.0, 31.4 (C-2), 58.2,58.4, 61.8, 62.0 (C-6′), 62.6 (C-5″), 67.9, 68.2, 70.8, 71.2, 73.8,74.7, 77.9, 79.5, 80.4, 96.4 (C-1′), 107.6 (C-1″), 128.4 (2C), 128.6(3C), 128.7, 128.9, 129.7 (3C), 129.8 (3C), 130.3 (2C), 133.3, 133.5,133.7, 165.2, 165.4, 166.2, 169.7, 170.1 (2C), 170.7.

MALDI TOFMS calculated for C₄₆H₄₇N₉O₁₈ K ([M+K]⁺) m/e: 1052.3; measuredm/e: 1052.4.

Preparation of Compound 16b

Anhydrous CH₂Cl₂ (5 ml) was added to powdered, flame-dried 4 Å molecularsieves (500 mg), followed by the addition of the acceptor Compound 11(300 mg, 0.527 mmol) and the donor Compound 15b (1 gram, 1.896 mmol),both of which were prepared as presented hereinabove. The reactionmixture was stirred for 10 minutes at room temperature, and then cooledto −40° C. Thereafter a catalytic amount of BF₃-Et₂O (10 μl) was addedto the reaction mixture and stirring continued at −15° C. Propagation ofthe reaction was monitored by TLC (EtOAc/Hexane, 3:7), which indicatedcompletion after 1.5 hours. The reaction mixture was diluted withCH₂Cl₂, and filtered through celite. After thorough washing of thecelite with CH₂Cl₂, the washes were combined and extracted withsaturated aqueous NaHCO₃, brine, dried over MgSO₄ and concentrated. Thecrude product was purified by flash chromatography to yield Compound 16b(350 mg, yield of 71%).

¹H NMR (500 MHz, CDCl₃) data of Compound 16b are summarized in Table 3below.

TABLE 3 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.84 d 3.56-3.59 m 5.41 t 5.07t 4.52-4.55 m 4.15-4.18 m 4.25-4.29 m J = 4.0 J = 9.5 J = 9.5 III 5.66 s5.58 d 5.39-5.46 m 4.49-4.55 m 3.56-3.61 m 3.56-3.61 m J = 4.5 H1 H2eqH2ax H3 H4 H5 H6 II 3.49-3.55 m 2.41 dt 1.61 ddd 3.49-3.55 m 3.73 t 3.88t 5.02 t J = 5.0, 12.5 J₁ = J₂ = J = 9.5 J = 9.5 J = 10.0 J₃ = 12.5

Additional ¹H NMR (500 MHz, CDCl₃) data for Compound 16b included:δ=2.05 (s, 3H, Ac), 2.09 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.31 (s, 3H,Ac), 7.34-7.39 (m, 2H, Bz), 7.40-7.43 (m, 2H, Bz), 7.52-7.61 (m, 2H,Bz), 7.88 (d, 2H, J=8, Bz), 7.95 (d, 2H, J=8.5, Bz).

¹³C NMR (125 MHz, CDCl₃): δ=20.6, 20.7, 20.8, 20.9, 31.7 (C-2), 52.1(C-5″), 58.3, 58.6, 61.7, 61.8 (C-6′), 68.1, 68.2, 70.8, 71.0, 73.9,74.6, 77.8, 79.8, 80.8, 96.7 (C-1′), 107.6 (C-1″), 128.5 (3C), 128.6(2C), 128.8, 129.6 (4C), 133.6, 133.7, 165.2, 165.4, 169.8, 170.0 (2C),170.7. MALDI TOFMS calculated for C₃₉H₄₂N₁₂O₁₆ Na ([M+Na]⁺) m/e 957.3;measured m/e 957.5.

Preparation of Compound 17a

Anhydrous CH₂Cl₂ (5 ml) was added to powdered, flame-dried 4 Å molecularsieves (800 mg), followed by the addition of the acceptor Compound 13(420 mg, 0.674 mmol) and the donor Compound 14a (334 mg, 0.808 mmol),both of which were prepared as presented hereinabove. The reactionmixture was stirred for 10 minutes at room temperature, and thenN-iodosuccinimide (NIS, 290 mg, 0.129 mmol) was added to the reactionmixture and stirring continued at room temperature for 5 minutes.Thereafter the reaction mixture was cooled to −40° C. and a catalyticamount of TfOH (10 μl) was added thereto. Propagation of the reactionwas monitored by TLC (EtOAc/Hexane, 2:3), which indicated completionafter 2 hours. The reaction mixture was diluted with CH₂Cl₂, andfiltered through celite. After thorough washing of the celite withEtOAc, the washes were combined and extracted with Na₂S₂O₃ (10%),saturated aqueous NaHCO₃, brine, dried over MgSO₄ and concentrated underreduced pressure. The crude product was purified by flash chromatographyto yield Compound 17a (490 mg, yield of 68%).

¹H NMR (500 MHz, CDCl₃) data of Compound 17a are summarized in Table 4below.

TABLE 4 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.42 d 3.72-3.75 m 5.89 t4.31-4.36 m 3.79 m 3.76-3.79 m 4.31-4.36 m J = 4.0 J = 10.0 J = 9.5 III5.78 s 5.78 s 5.84 t 4.77-4.80 m 4.77-4.80 m 4.69 dd J = 5.5 J = 7.0,12.5 H1 H2eq H2ax H3 H4 H5 H6 II 3.31-3.43 m 2.33-2.37 m 1.46-1.54 m3.31-3.43 m 3.31-3.43 m 3.71-3.75 m 3.59 t J = 9.5

Additional ¹H NMR (500 MHz, CDCl₃) data for Compound 17a included:δ=5.49 (s, 1H), 7.32-7.61 (m, 14H), 7.89 (d, 2H, J=7.5 Hz), 7.92 (d, 2H,J=7.0 Hz), 7.99 (d, 2H, J=7.0 Hz), 8.07-8.09 (m, 4H).

¹³C NMR (125 MHz, CDCl₃): δ=32.0 (C-2), 55.2 (CH₃), 58.2, 58.8, 62.7,63.5, 65.1 (C-6′), 68.6 (C-5″), 70.4, 72.3, 75.7, 76.1, 79.1, 79.3,80.8, 82.9, 99.8 (C-1′), 101.5, 106.5 (C-1″), 113.5 (2C), 127.4-133.5(27C), 160.0, 165.3 (2C), 165.6, 166.1.

MALDI TOFMS calculated for C₅₃H₄₉N₉O₁₆ Na ([M+Na]⁺) m/e: 1090.3;measured m/e: 1090.3.

Preparation of Compound 17b

Anhydrous CH₂Cl₂ (5 ml) was added to powdered, flame-dried 4 Å molecularsieves (500 mg), followed by the addition of the acceptor Compound 13(350 mg, 0.561 mmol) and the donor Compound 14b (334 mg, 0.682 mmol),both of which were prepared as presented hereinabove. The reactionmixture was stirred for 10 minutes at room temperature, and then NIS(290 mg, 0.129 mmol) was added to the reaction mixture and stirringcontinued at room temperature for 5 minutes. Thereafter the reactionmixture was cooled to −40° C. and a catalytic amount of TfOH (10 μl) wasadded thereto. Propagation of the reaction was monitored by TLC(EtOAc/Hexane, 2:3), which indicated completion after 2 hours. Thereaction mixture was diluted with CH₂Cl₂, and filtered through celite.After thorough washing of the celite with EtOAc, the washes werecombined and extracted with Na₂S₂O₃ (10%), saturated aqueous NaHCO₃,brine, dried over MgSO₄ and concentrated under reduced pressure. Thecrude product was purified by flash chromatography to yield Compound 17bas a mixture of anomers at a ratio of α/β 1:5 (420 mg, yield of 76%).

¹H NMR (500 MHz, CDCl₃) data of Compound 17b are summarized in Table 5below.

TABLE 5 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.39 d 3.70-3.73 m 5.59 t4.10-4.13 m 3.88 t 3.90-3.92 m 3.90-3.92 m J = 3.5 J = 10.0 J = 9.5 III5.80 s 5.71 d 5.62-5.65 m 4.54-4.56 m 3.63-3.65 m 3.74-3.79 m J = 5.0 H1H2eq H2ax H3 H4 H5 H6 II 3.33-3.40 m 2.332.37 m 1.49-1.57 m 3.45-3.50 m3.33-3.40 m 3.74-3.79 m 3.62 t J = 8.5

Additional ¹H NMR (500 MHz, CDCl₃) data for Compound 17b included:δ=5.50 (s, 1H), 7.34 (t, 2H, J=7.5 Hz), 7.39 (t, 2H, J=7.5 Hz), 7.45 (t,2H, J=7.5 Hz), 7.51-7.61 (m, 3H), 7.89 (d, 2H, J=7.5 Hz), 7.98 (d, 2H,J=7.5 Hz), 8.07 (d, 2H, J=7.5 Hz).

¹³C NMR (125 MHz, CDCl₃): δ=31.9 (C-2), 53.1 (C-5″), 55.1, 58.7, 62.7,63.4, 68.5 (C-6′), 70.4, 72.2, 75.6, 76.1, 79.1 (2C), 83.1, 99.9 (C-1′),101.4, 105.9 (C-1″), 113.4 (2C), 127.3-133.5 (21C), 159.9, 165.2 (2C),165.3.

MALDI TOFMS calculated for C₄₆H₄₄N₁₂O₁₄ Na ([M+Na]⁺) m/e: 1011.3;measured m/e: 1011.6.

Preparation of Compound 18a

Anhydrous CH₂Cl₂ (5 ml) was added to powdered, flame-dried 4 Å molecularsieves (500 mg), followed by the addition of the acceptor Compound 12(200 mg, 0.356 mmol) and the donor Compound 15a (300 mg, 0.494 mmol),both of which were prepared as presented hereinabove. The reactionmixture was stirred for 10 minutes at room temperature, and then cooledto −40° C. Thereafter a catalytic amount of BF₃-Et₂O (10 μl) was addedto the reaction mixture and stirring continued at −20° C. Propagation ofthe reaction was monitored by TLC (EtOAc/Hexane, 15:85), which indicatedcompletion after 3 hours. The reaction mixture was diluted with CH₂Cl₂,and filtered through celite. After thorough washing of the celite withCH₂Cl₂, the washes were combined and extracted with saturated aqueousNaHCO₃, brine, dried over MgSO₄ and concentrated. The crude product waspurified by flash chromatography to yield Compound 18a (340 mg, yield of95%).

¹H NMR (500 MHz, CDCl₃) data of Compound 18a are summarized in Table 6below.

TABLE 6 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.12 d 3.35-3.37 m 3.91-3.96 m3.51-3.54 m 3.91-3.96 m 3.75-3.83 m 3.75-3.83 m J = 3.5 III 5.55 s 5.75d 5.87 t 4.77-4.81 m 4.57 dd 4.93 dd J = 4.5 J = 5.0 J = 5.0, 12.5 J =4.0, 12.5 H1 H2eq H2ax H3 H4 H5 H6 II 3.23-3.28 m 2.30 dt 1.47 ddd3.38-3.52 m 3.23-3.28 m 3.38-3.52 m 3.38-3.52 m J = 4.0, 13.5 J₁ = J₂ =J₃ = 12.5

Additional ¹H NMR (500 MHz, CDCl₃) data for Compound 18a included:δ=1.25-1.30 (m, 5H), 1.62-1.89 (m, 15H), 7.34-7.61 (m, 9H, aromatic),7.92 (d, 2H, J=7.5 Hz), 8.02 (d, 2H, J=7.5 Hz), 8.11 (d, 2H, J=7.5 Hz).

¹³C NMR (125 MHz, CDCl₃): δ=24.7 (2C), 26.8 (4C), 31.9 (C-2), 41.8 (4C),52.6, 58.6, 59.4, 61.9 (C-5″), 62.5, 63.6 (C-6′), 69.2, 71.4, 71.8,75.1, 75.2, 75.7, 76.0, 81.2, 83.6, 85.0, 98.8 (C-1′), 107.2 (C-1″),128.3-129.7 (15C), 133.4, 133.5 (2C), 165.0, 165.2, 166.1.

ESIMS calculated for C₅₀H₅₅N₉O₁₄ Na ([M+Na]⁺) m/e: 1044.4; measured m/e:1044.4.

Preparation of Compound 18b

Anhydrous CH₂Cl₂ (5 ml) was added to powdered, flame-dried 4 Å molecularsieves (500 mg), followed by the addition of the acceptor Compound 12(340 mg, 0.605 mmol) and the donor Compound 15b (600 mg, 1.107 mmol),both of which were prepared as presented hereinabove. The reactionmixture was stirred for 10 minutes at room temperature, and then cooledto −20° C. Thereafter a catalytic amount of BF₃-Et₂O (10 μl) was addedto the reaction mixture and stirring continued at −20° C. Propagation ofthe reaction was monitored by TLC (EtOAc/Hexane, 15:85), which indicatedcompletion after 3 hours. The reaction mixture was diluted with CH₂Cl₂,and filtered through celite. After thorough washing of the celite withCH₂Cl₂, the washes were combined and extracted with saturated aqueousNaHCO₃, brine, dried over MgSO₄ and concentrated. The crude product waspurified by flash chromatography to yield Compound 18b (520 mg, yield of93%).

¹H NMR (500 MHz, CDCl₃) data of Compound 18b are summarized in Table 7below.

TABLE 7 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.51 d 3.36-3.39 m 4.22 t 3.99ddd 3.73-3.79 m 3.73-3.80 m 3.53 dd J = 3.5 J = 10.5 J = 5.0, J = 5.0,10.5 10.5, 15.0 III 5.70 S 5.64-5.69 m 5.64-5.69 m 4.52 ddd 3.87 dd 3.53dd J = 3.5, J = 7.0, 13.0 J = 3.5, 13.0 10.5, 14.5 H1 H2eq H2ax H3 H4 H5H6 II 3.52 ddd 2.36 dt 1.61 ddd 3.66 ddd 3.40 t 3.50-3.55 m 3.76-3.80 mJ = 4.5, J = 5.0, 13.5 J₁ = J₂ = J = 4.5, J = 9.5 10.0, 14.0 J₃ = 12.511.0, 15.0

Additional ¹H NMR (500 MHz, CDCl₃) data for Compound 18b included:δ=7.33 (t, 2H, J=7.5 Hz, Bz), 7.44 (t, 2H, J=7.5 Hz, Bz), 7.52 (t, 1H,J=7.5 Hz, Bz), 7.59 (t, 2H, J=7.5 Hz, Bz), 7.87 (d, 2H, J=7.5 Hz, Bz),8.03 (d, 2H, J=7.5 Hz, Bz).

¹³C NMR (125 MHz, CDCl₃): δ=(C from 22.4-37.8 from cyclohexanones) 22.4,22.6, 23.7 (2C), 24.8, 25.5, 27.9, 33.9 (C-2), 36.0, 36.3, 37.8, 51.9(C-5″), 57.2, 60.5, 61.6 (C-6′), 63.2, 64.4, 72.2, 72.3, 75.5, 79.2,79.3, 80.8, 83.1, 84.1, 97.0 (C-1′), 100.3 (OCO cyclohexanone ketal),105.0 (C-1″), 113.7 (OCO cyclohexanone ketal), 128.4-133.8 (12C), 165.2,165.4.

MALDI TOFMS calculated for C₄₃H₅₀N₁₂O₁₂ Na ([M+Na]⁺) m/e: 949.4;measured m/e: 949.3.

Preparation of Compound 19a

Compound 12 (300 mg, 0.534 mmol) was dissolved in CH₂Cl₂ (3 ml) andadded to dry pyridine (5 ml). The reaction mixture was stirred for 5minutes at room temperature, cooled to −15° C. in an ice bath, andtriflic anhydride (Tf₂O, 300 mg, 1.067 mmol) was added thereto drop wiseover 5 minutes. The ice bath was removed after 15 minutes and thereaction mixture was heated to room temperature. Propagation of thereaction was monitored by TLC (EtOAc/Hexane 1:4), which indicatedcompletion after 1.5 hours. The reaction was diluted with CH₂Cl₂, andextracted with saturated aqueous NaHCO₃, HCl (2%) and brine. The organiclayer was dried over MgSO₄ and concentrated under reduced pressure. Thecrude product was purified by flash chromatography to yield Compound 19a(340 mg, yield of 92%).

¹H NMR (500 MHz, CDCl₃): δ=1.25-1.71 (m, 20H, cyclohexanones), 1.51(ddd, 1H, J₁=J₂=J₃=12.5 Hz, H-2 axial), 2.39 (dt, 1H, J₁=5.0, J₂=14.0H-2equatorial), 3.36 (dd, 1H, J₁=4.0 J₂=10.0 Hz, H-2′), 3.41 (t, J=9.5 Hz,1H, H-4), 3.51-3.55 (m, 1H, H-3), 3.58 (t, 1H, J₁=9.5, J₂=10.0 Hz, H-5),3.66-3.70 (m, 1H, H-1), 3.80 (t, 1H, J=9.5 Hz, H-6), 3.78-3.84 (m, 2H,H-5′, H-6′), 3.96 (dd, 1H, J₁=5.0 J₂=11.0 Hz, H-6′), 4.00-4.05 (m, 1H,H-4′), 5.07 (t, 1H, J=10.0 Hz, H-3′), 5.63 (d, 1H, J=3.5 Hz, H-1′).

¹³C NMR (125 MHz, CDCl₃): δ=(the range 22.4-37.6 relates to cyclohexanerings carbon atoms if not otherwise indicated) 22.1, 22.2, 23.7 (2C),24.8, 25.5, 27.7, 33.7 (C-2), 36.0, 36.3, 37.6, 57.2, 60.2, 61.0, 61.3(C-6′), 64.4, 70.9, 77.7, 78.1, 78.3, 79.2, 83.2 (C-3′), 97.7 (C-1′),100.7 (OCO cyclohexanone ketal), 114.0 (OCO cyclohexanone ketal),

ESIMS calculated for C₂₅H₃₄F₃N₉O₉SNa ([M+Na]⁺) m/e: 716.2; measured m/e:716.2.

Preparation of Compound 19

Compound 19a (330 mg, 0.476 mmol), prepared as presented hereinabove,was dissolved in DMF (2 ml) and hexamethylphosphoramide (HMPA, 1 ml),followed by the addition of NaN₃ (310 mg, 4.77 mmol), and the reactionmixture was stirred at 80° C. Propagation of the reaction was monitoredby TLC (EtOAc/Hexane, 15:85), which indicated completion after 2 hours.The reaction was diluted with CH₂Cl₂, extracted with brine, dried overMgSO₄ and concentrated under reduced pressure. The crude product waspurified by flash chromatography to yield Compound 19 (200 mg, yield of72%).

¹H NMR (500 MHz, CDCl₃) data of Compound 19 are summarized in Table 8below.

TABLE 8 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.44 d 3.17 t 4.18 t 4.30-4.36m 3.72-3.78 m 3.72-3.78 m 3.97 dd J = 4.0 J = 4.0 J = 3.5 J = 6.0, 11.0H1 H2eq H2ax H3 H4 H5 H6 II 3.63-3.66 m 2.36 dt 1.48 ddd 3.53-3.57 m3.38 t 3.59 t 3.75 t J = 5.0, 13.5 J₁ = J₂ = J = 9.5 J = 9.5 J = 9.5 J₃= 12.5

Additional ¹H NMR (500 MHz, CDCl₃) data for Compound 19 included:δ=1.26-1.93 (m, 20H, cyclohexane rings).

¹³C NMR (125 MHz, CDCl₃): δ=(the range 22.6-37.6 relates to cyclohexanerings carbon atoms if not otherwise indicated) 22.6 (2C), 23.7 (2C),24.9, 25.5, 27.8, 34.2 (C-2), 35.9, 36.3, 37.6, 56.9, 57.3, 59.8, 60.4,60.8, 61.4 (C-6′), 70.9, 77.1, 79.3 (2C), 96.5 (C-1′), 100.0 (OCOcyclohexanone ketal), 113.6 (OCO cyclohexanone ketal).

ESIMS calculated for C₂₄H₃₄N₁₂O₆ Na ([M+Na]⁺) m/e: 609.3; measured m/e:609.3.

Preparation of Compound 19c

Compound 19 (200 mg, 0.352 mmol), prepared as presented hereinabove, wasdissolved in dioxane (3 ml) and added to acetic acid (8 ml) and water (1ml), and the reaction mixture was stirred at 75° C. Propagation of thereaction was monitored by TLC (EtOAc/Hexane, 7:3), which indicatedcompletion after 3 hours. The reaction mixture was diluted with EtOAc,washed with saturated aqueous NaHCO₃ and brine. The combined organiclayer was dried over MgSO₄, evaporated under reduced pressure andpurified by flash chromatography (silica, EtOAc/Hexane) to yieldCompound 19c (90 mg, yield of 60%).

¹H NMR (500 MHz, MeOD): δ=1.41 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H-2 axial),2.39 (dt, 1H, J₁=4.5, J₂=13.0 Hz, H-2 equatorial), 3.27 (t, 1H, J=9.0Hz, H-4), 3.33 (t, 1H, J=4.0 Hz, H-2′), 3.35-3.50 (m, 3H, H-1, H-3 andH-6), 3.43 (d, 1H, J=3.5 Hz, H-4′), 3.54 (t, 1H, J=9.5 Hz, H-5′), 3.80(t, 1H, J=9.5 Hz, H-6), 3.79-3.81 (m, 2H, H-6′), 4.14-4.16 (m, 1H,H-3′), 5.55 (d, 1H, J=4.0 Hz, H-1′).

¹³C NMR (125 MHz, CDCl₃): δ=31.8 (C-2), 57.2, 59.2, 59.7, 60.3 (C-6′),63.3, 66.2, 67.3, 75.8, 75.9, 78.8, 96.5 (C-1′).

MALDI TOFMS calculated for C₁₂H₁₉N₁₂O₆ ([M+H]⁺) m/e: 427.3; measuredm/e: 427.3.

Preparation of Compound 20

Dry mixture of DMF/HMPA (2:1, 3 ml) was added to powdered, flame-dried 4Å molecular sieves (500 mg), followed by the addition of thedibromomethane (19 μl, 0.26 mmol) and the acceptor Compound 12 (290 mg,0.516 mmol) which was prepared as presented hereinabove. The reactionmixture was stirred for 10 minutes at room temperature, cooled to −10°C., and then NaH (19 mg, 0.792 mmol) was added thereto. After 15 minutesof stirring the reaction mixture was heated to 40° C. Propagation of thereaction was monitored by TLC (EtOAc/Hexane, 15:85), which indicated thecompletion after 2 hours. The reaction was diluted with EtOAc, andfiltered through celite. After thorough washing of the celite withEtOAc, the washes were combined and extracted with brine, dried overMgSO₄ and concentrated under reduced pressure. The crude was purified byflash chromatography to yield Compound 20 (240 mg, yield of 82%).

¹H NMR (500 MHz, CDCl₃) data of Compound 20 are summarized in Table 9below.

TABLE 9 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.49 d 3.33 dd 4.09 t 3.63-3.67m 3.88-3.93 m 3.75-3.80 m 3.88-3.93 m J = 8.5 J = 3.0, 14.0 J = 4.5 H1H2eq H2ax H3 H4 H5 H6 II 3.62-3.67 m 2.33 dt 1.47 ddd 3.43-3.46 m 3.39 t3.53 t 3.75-3.80 m J = 5.0, 13.5 J₁ = 12 J = 10 J = 9.5 J₂ = J₃ = 13.0

Additional ¹H NMR (500 MHz, CDCl₃) data for Compound 20 included:δ=1.23-1.70 (m, 40H, cyclohexane rings), 5.21 (s, 2H, H-1″).

¹³C NMR (125 MHz, CDCl₃): δ=(the range 22.4-37.6 relates to cyclohexanerings carbon atoms if not otherwise indicated) 22.4, 23.5 (2C), 24.7,25.4, 27.5 (C-2), 33.7, 35.8, 36.1, 37.6, 57.0, 60.4, 61.5 (C-6′), 62.1,63.9, 73.6, 74.1, 78.0, 78.2, 96.6 (C-1″ half of other), 97.0 (C-1′),99.6 (OCO cyclohexanone ketal), 115.4 (OCO cyclohexanone ketal).

MALDI TOFMS calculated for C₄₉H₇₀N₁₈O₁₄ Na ([M+Na]⁺) m/e: 1157.5;measured m/e: 1157.4.

Preparation of Compound 20c

Compound 20 (240 mg), prepared as presented hereinabove), was dissolvedin THF (5 ml) and added to a mixture of TFA (1 ml) and water (1.2 ml).The reaction mixture was stirred at 60° C. for 2 hours. Propagation ofthe reaction was monitored by TLC (EtOAc/Hexane, 9:1). The reactionmixture was purified by flash chromatography (silica, EtOAc/Hexane) toyield Compound 20c (155 mg, yield of 90%).

¹H NMR (500 MHz, MeOD): δ=□□1.40 (ddd, 1H, J₁=12.5, J₂=J₃=12 Hz, H-2axial), 2.23 (dt, 1H, J₁=5 J₂=13.5 Hz, H-2 equatorial), 3.27-3.43 (m,4H, H-1, H-3, H-4 and H-5), 3.38-3.48 (m, 1H, H-2′), 3.50 (t, 1H, J₁=8.5Hz, H-6), 3.59 (t, 1H, J₁=7 Hz, H-5′), 3.76-3.83 (m, 2H, H-4′ and H-6′),3.88-3.95 (m, 1H, H-6′), 3.91 (t, 1H, J=9.5 Hz, H-3′), 5.16 (s, 1H,H-1″), 5.49 (d, 1H, J=3.5 Hz, H-1′).

¹³C NMR (125 MHz, MeOD): δ=31.3 (C-2), 61.0, 61.8, 62.8 (C-6′), 65.1,70.9, 74.2, 77.7, 78.0, 82.1, 82.2, 99.9 (C-1″ tether carbon), 99.9(C-1′).

MALDI TOFMS calculated for C₂₅H₃₈N₈O₁₄ Na ([M+Na]⁺) m/e: 837.3; measuredm/e: 837.2.

Preparation of Compound 2

Compound 2 was prepared following the chart presented in FIG. 10,starting from Compound 10 which was converted into Compound 11 andcoupled to Compound 15a to afford Compound 16a as described hereinabove.

Compound 16a (450 mg, 0.444 mmol), prepared as presented hereinabove,was treated with a solution of MeNH₂ (33% solution in 30 ml EtOH) andthe propagation of the reaction was monitored by TLC (EtOAc/MeOH, 7:3),which indicated completion after 8 hours. The reaction mixture wasevaporated to dryness under reduced pressure and the residue wasdissolved in a mixture of THF (5 ml) and aqueous NaOH (0.1 M, 3.5 ml).This mixture was stirred at room temperature for 10 minutes andthereafter PMe₃ (1 M solution in THF, 2.66 ml THF, 2.66 mmol) was addedthereto. Propagation of the reaction was monitored by TLC, using amixture of CH₂Cl₂/MeOH/H₂O/MeNH₂ at a relative ratio of 10:15:6:15diluted to 33% solution in ethanol as eluent, which indicated completionafter 5 hours.

The reaction mixture was purified by flash chromatography on a shortcolumn of silica gel. The column was washed with the following solvents:THF (100 ml), CH₂Cl₂ (200 ml), EtOH (100 ml), and MeOH (150 ml). Theproduct was eluted with the mixture of MeNH₂ (33% solution in EtOH) andMeOH at a ratio of 1:4. Fractions containing the product were combinedand evaporated under reduced pressure, re-dissolved in small volume ofwater and evaporated again under reduced pressure. This procedure wasrepeated 2 to 3 times to afford the free amine form of Compound 2 (170mg, yield of 84%). This product was dissolved in water, the pH wasadjusted to 6.5 by H₂SO₄ (0.01 M) and lyophilized to afford the sulfatesalt of Compound 2.

¹H NMR (500 MHz, D₂O, pH=3.5) data of Compound 2 are summarized in Table10 below.

TABLE 10 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.66 d 3.26-3.29 m 3.83 t3.70-3.73 m 3.35 t 3.79-3.83 m 3.66-3.71 m J = 4.0 J = 10.0 J = 9.0 III5.22 s 4.08-4.09 m 4.08-4.09 m 3.90-3.92 m 3.77-3.80 m 3.60 dd J = 5.0,13.0 H1 H2eq H2ax H3 H4 H5 H6 II 3.18-3.23 m 2.30 dt 1.65 ddd 3.33-3.39m 3.56 t 3.75-3.81 m 3.77-3.81 m J = 4.5, 12.5 J₁ = J₂ = J = 9.0 J₃ =12.5

¹³C NMR (125 MHz, D₂O): δ=□□31.1 (C-2), 50.7, 51.8, 55.8, 62.1 (C-5″),62.5 (C-6′), 70.7, 70.8, 71.1, 74.4, 75.3, 76.9, 80.4, 84.1, 86.3, 97.8(C-1′), 111.9 (C-1″);

MALDI TOFMS calculated for C₁₇H₃₃N₃O₁₁ Na ([M+Na]⁺) m/e: 478.2; measuredm/e: 478.2.

Preparation of Compound 3

Compound 3 was prepared following the chart presented in FIG. 11,starting from Compound 10 and Compound 14b which were converted intoCompound 11 and Compound 15b respectively and coupled to one another toafford Compound 16b as described hereinabove.

Compound 16b (320 mg, 0.342 mmol), prepared as presented hereinabove,was treated with a solution of MeNH₂ (33% solution in 30 ml EtOH) andthe propagation of the reaction was monitored by TLC (EtOAc/MeOH, 7:3),which indicated completion after 8 hours. The reaction mixture wasevaporated to dryness under reduced pressure and the residue wasdissolved in a mixture of THF (3.7 ml) and aqueous NaOH (0.1 M, 2.5 ml).This mixture was stirred at room temperature for 10 minutes andthereafter PMe₃ (1 M solution in THF, 2.74 ml, 2.74 mmol) was addedthereto. Propagation of the reaction was monitored by TLC, using amixture of CH₂Cl₂/MeOH/H₂O/MeNH₂ at a relative ratio of 10:15:6:15diluted to 33% solution in ethanol as eluent, which indicated completionafter 5 hours.

The product was purified as described above for Compound 2 to yieldCompound 3 as a free amine (142 mg, yield of 91%).

¹H NMR (500 MHz, D₂O, pH=3.5) data of Compound 3 are summarized in Table11 below.

TABLE 11 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.75 d 3.40-3.46 m 3.94 m3.71-3.73 m 3.40-3.46 m 3.70-3.74 m 3.82-3.85 m J = 4.0 J = 9.5 III 5.31s 4.17-4.19 m 4.15 t 4.03-4.08 m 3.17 dd 3.28-3.32 m J = 10.0 J = 7.5,13.5 H1 H2eq H2ax H3 H4 H5 H6 II 3.28-3.32 m 2.41 dt 1.83 ddd 3.52-3.55m 3.69 t 3.93 t 4.06 t J = 4.0, 12.5 J₁ = J₂ = J = 9.5 J = 9.0 J = 9.0J₃ = 12.5

¹³C NMR (125 MHz, D₂O): δ□□=029.5 (C-2), 43.4 (C-5″), 50.9, 51.7, 55.3,61.9 (C-6′), 70.8 (2C), 72.8, 73.4, 75.9, 76.4, 77.8, 80.1, 84.2, 95.7(C-1′), 110.4 (C-1″).

MALDI TOFMS calculated for C₁₇H₃₃N₄O₁₀ Na ([M+Na]⁺) m/e 477.2; measuredm/e 477.5.

Preparation of Compound 4

Compound 4 was prepared following the chart presented in FIG. 12,starting from Compound 10 which was converted into Compound 13 which wascoupled to Compound 14a to afford Compound 17a as described hereinabove.

Compound 17a (460 mg, 0.43 mmol) was dissolved in THF (3 ml) and addedwith acetic acid (4.5 ml) and water (0.75 ml). The reaction mixture wasstirred at 50° C. for 3 hours and the propagation was monitored by TLC(EtOAc/Hexane, 3:2). The reaction mixture was diluted with EtOAc andwashed with saturated NaHCO₃ and brine. The combined organic layer wasdried over MgSO₄ and the mixture was evaporated to dryness under reducedpressure.

The residue was dissolved in THF (4 ml) and was added with the solutionof NaOH (0.1 M, 3 ml). The mixture was stirred at room temperature for10 minutes, after which PMe₃ (1 M solution in THF, 2.85 ml, 2.85 mmol)was added. Propagation of the reaction was monitored by TLC, using amixture of CH₂Cl₂/MeOH/H₂O/MeNH₂ at a relative ratio of 10:15:6:15diluted to 33% solution in ethanol as eluent, which indicated completionafter 5 hours. The reaction mixture was purified by flash chromatographyon a short column of silica gel. The column was washed with thefollowing solvents: THF (100 ml), CH₂Cl₂ (200 ml), EtOH (100 ml), andMeOH (150 ml). The product was eluted with the mixture of MeNH₂ (33%solution in EtOH) and MeOH at a ratio of 1:4. The fractions containingthe product were evaporated under reduced pressure, redissolved in waterand evaporated again, and the procedure was repeated 2 to 3 times toafford Compound 4 in free amine form (147 mg, overall yield of 75%). Theproduct was dissolved in water, the pH was adjusted to 6.65 with H₂SO₄(0.01 M) and lyophilized to afford the sulfate salt of Compound 4 as ayellow foamy solid.

¹H NMR (500 MHz, D₂O, pD 3.0, adjusted by H₂SO₄ 0.01M) data of Compound4 are summarized in Table 12 below.

TABLE 12 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.61 d 3.28 dd 3.84 t3.70-3.73 m 3.36 t 3.80-3.87 m 3.64-3.71 m J = 4.0 J = 4.0, 10.5 J =10..0 J = 9.5 III 5.07 s 4.11 d 4.23-4.26 m 3.91-3.94 m 3.65-3.69 m3.74-3.78 m J = 5.0 H1 H2eq H2ax H3 H4 H5 H6 II 3.22-3.28 m 2.43 dt 1.77ddd 3.42-3.47 m 3.58 t 3.80 t 3.84 t J = 4.0, 12.0 J₁ = J₂ = J = 9.5 J =9.5 J = 9.5 J₃ = 12.5

¹³C NMR (125 MHz, D₂O): δ=□□29.8 (C-2), 49.9, 50.5, 55.8, 60.7 (C-5″),62.1 (C-6′), 70.3, 70.7, 71.2, 75.4, 76.0, 76.4, 81.1, 82.4, 84.1, 98.5(C-1′), 110.5 (C-1″).

MALDI TOFMS calculated for C₁₇H₃₃N₄O₁₀ Na ([M+Na]⁺) m/e: 478.2; measuredm/e: 478.2.

Preparation of Compound 5d

Compound 17b (400 mg, 0.40 mmol) was dissolved in THF (3 ml) and add toa mixture of TFA (1.5 ml) and water (1 ml). The reaction mixture wasstirred at 50° C. for 2 hours during which the propagation was monitoredby TLC (EtOAc/Hexane, 1:1). The reaction mixture was directly applied ona silica-gel column and purified by flash chromatography (EtOAc/Hexane)to yield Compound 5d (180 mg, yield of 51.6%).

¹H NMR (500 MHz, CDCl₃): δ=□□1.49-1.57 (m, 1H, H-2 axial), 2.33-2.37 (m,1H, H-2 equatorial), 3.33-3.40 (m, 2H, H-3, H-4), 3.45-3.50 (m, 1H,H-1), 3.62 (t, 1H, J=8.5 Hz, H-6), 3.63-3.65 (m, 1H, H-5″), 3.70-3.73(m, 1H, H-2′), 3.74-3.79 (m, 2H, H-5 and H-5″), 3.88 (t, 1H, J=9.5 Hz,H-5′), 3.90-3.92 (m, 2H, H-6′), 4.10-4.13 (m, 1H, H-4′), 4.53-4.56 (m,1H, H-4″), 5.39 (d, 1H, J=3.5 Hz, H-1′), 5.59 (t, 1H, J=10 Hz, H-3′),5.62-5.65 (m, 1H, H-3″), 5.71 (d, 1H, J=5 Hz, H-2″), 5.80 (s, 1H, H-1″),7.34 (t, 2H, J=7.5 Hz), 7.39 (t, 2H, J=7.5 Hz), 7.45 (t, 2H, J=7.5 Hz),7.51-7.61 (m, 3H), 7.89 (d, 2H, J=7.5 Hz), 7.98 (d, 2H, J=7.5 Hz), 8.07(d, 2H, J=7.5 Hz).

¹³C NMR (125 MHz, CDCl₃): δ=32.2 (C-2), 53.4 (C-5″), 58.5, 58.9, 61.6(C-6′), 62.3, 69.5, 72.4, 72.6, 75.0, 75.8, 76.3, 79.2, 79.3, 83.2, 99.1(C-1′), 106.2 (C-1″), 128.4-130.0 (15C), 133.6 (2C), 133.7, 165.5 (2C),167.2.

MALDI TOFMS calculated for C₃₈H₃₈N₁₂O₁₃ Na ([M+Na]⁺) m/e: 893.3;measured m/e: 893.2.

Preparation of Compound 5

Compound 5 was prepared following the chart presented in FIG. 13,starting from Compound 10 that was converted into Compound 13, which wascoupled to Compound 14b to afford Compound 17b, that was converted intoCompound 5d as described hereinabove.

Compound 5d (140 mg, 0.16 mmol) was dissolved in THF (3 ml) and wasadded to a solution of NaOH (0.1M, 2 ml). The reaction mixture wasstirred at room temperature for 10 minutes, and thereafter PMe₃ (1Msolution in THF, 1.65 ml, 1.65 mmol) was added thereto. Propagation ofthe reaction was monitored by TLC, using a mixture ofCH₂Cl₂/MeOH/H₂O/MeNH₂ at a relative ratio of 10:15:6:15 diluted to 33%solution in ethanol as eluent, which indicated completion after 5 hours.The reaction mixture was purified by flash chromatography on a shortcolumn of silica gel. The column was washed with the following solvents:THF (100 ml), CH₂Cl₂ (200 ml), EtOH (100 ml), and MeOH (150 ml). Theproduct was eluted with a mixture of MeNH₂ (33% solution in EtOH, 30 ml)and MeOH at a ratio of 1:4. The fractions containing the product wereevaporated under reduced pressure, redissolved in water and evaporatedunder reduced pressure. This procedure was repeated 2 to 3 times toafford Compound 5 in free amine form (32 mg, yield of 44%). The aminewas dissolved in water, the pH was adjusted to 6.5 with H₂SO₄ (0.01 M)and lyophilized to give the sulfate salt of Compound 5 as a yellow foamysolid.

¹H NMR (500 MHz, D₂O, pH=3.0) data of Compound 5 are summarized in Table13 below.

TABLE 13 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.57 d 3.25-3.36 m 3.80-3.84 m3.80-3.84 m 3.31-3.38 m 3.59-3.63 m 3.67-3.72 m J = 4.0 III 5.19 d3.97-4.06 m 3.97-4.06 m 3.97-4.06 m 3.05-3.10 m 3.26-3.30 m J = 1.5 H1H2eq H2ax H3 H4 H5 H6 II 3.31-3.38 m 2.41-2.44 m 1.83 ddd 3.42-3.48 m3.71-3.72 m 3.71-3.73 m 3.82-3.86 m J₁ = J₂ = J₃ = 12.5

¹³C NMR (125 MHz, D₂O): δ=□□29.7 (C-2), 43.9 (C-5″), 49.8, 50.5, 55.7,62.1 (C-6′), 70.7, 71.2, 72.8, 75.4, 75.8, 76.0, 79.9, 81.6, 81.8, 98.6(C-1′), 110.7 (C-1″);

TOF APMS calculated for C₁₇H₃₄N₄O₁₀ ([M+H]⁺) m/e: 455.2; measured m/e:455.2.

Preparation of Compound 6a

Compound 18a (240 mg, 0.24 mmol) was dissolved in dioxane (6 ml) andadded to a mixture of acetic acid (10 ml) and water (3 ml). The reactionmixture was stirred at 70° C. for 5 hours. Propagation of the reactionwas monitored by TLC (EtOAc/Hexane, 7:3). The reaction mixture wasdiluted with EtOAc and washed with saturated NaHCO₃ and brine. Thecombined organic layer was dried over MgSO₄, evaporated under reducedpressure and purified by flash chromatography (silica, EtOAc/Hexane) toyield Compound 6a (215 mg, yield of 75%).

¹H NMR (500 MHz, CDCl₃): δ=□1.47 (ddd, 1H, J₁=11.5 Hz, J₃=J₂=12.5 Hz,H-2 axial), 2.30 (dt, 1H, J₁=4 J₂=13 Hz, H-2 equatorial), 3.21-3.30 (m,2H, H-3, H-4), 3.35-3.37 (m, 1H, H-2′), 3.40-3.50 (m, 3H, H-1, H-5,H-6), 3.51-3.54 (m, 1H, H-4′), 3.75 (dd, 1H, J₁=4 J₂=12 Hz, H-6′), 3.82(dd, 1H, J₁=4 J₂=12 Hz, H-6′), 3.90-3.96 (m, 2H, H-3′, H-5′), 4.57 (dd,1H, J₁=5 J₂=12.5 Hz, H-5″), 4.78-4.81 (m, 1H, H-4″), 4.93 (dd, 1H, J₁=4J₂=12.5 Hz, H-5″), 5.11 (d, 1H, J=4 Hz, H-1′), 5.55 (s, 1H, H-1″), 5.76(d, 1H, J=5 Hz, H-2″), 5.87 (t, 1H, J=5 Hz, H-3″), 7.35-7.61 (m, 9H),7.92 (d, 2H, J=7.5 Hz), 8.02 (d, 2H, J=7.5 Hz), 8.12 (d, 2H, J=7.5 Hz).

¹³C NMR (125 MHz, CDCl₃): δ=31.9 (C-2), 52.6, 58.6, 59.4 (C-5″), 62.5,63.6 (C-6′), 69.2, 71.4, 71.8, 75.1, 75.2, 76.2, 80.1, 83.7, 85.0, 98.9(C-1′), 107.2 (C-1″), 128.3-129.7 (15C), 133.4, 133.5 (2C), 165.0,165.2, 166.1;

MALDI TOFMS calculated for C₃₈H₃₉N₉O₁₄ Na ([M+Na]⁺) m/e: 868.3; measuredm/e: 868.4.

Preparation of Compound 6

Compound 6 was prepared following the chart presented in FIG. 14,starting from Compound 10 that was converted into Compound 12, which wascoupled to Compound 15a to afford Compound 18a, that was converted intoCompound 6a as described hereinabove.

Compound 6a (200 mg, 0.236 mmol) was dissolved in THF (3 ml) and wasadded to a solution of NaOH (0.1M, 1.5 ml). The reaction mixture wasstirred at room temperature for 10 minutes, and thereafter PMe₃ (1Msolution in THF, 1.4 ml, 1.4 mmol) was added thereto. Propagation of thereaction was monitored by TLC, using a mixture of CH₂Cl₂/MeOH/H₂O/MeNH₂at a relative ratio of 10:15:6:15 diluted to 33% solution in ethanol aseluent, which indicated completion after 5 hours. The reaction mixturewas purified by flash chromatography on a short column of silica gel.The column was washed with the following solvents: THF (100 ml), CH₂Cl₂(200 ml), EtOH (100 ml), and MeOH (150 ml). The product was eluted witha mixture of MeNH₂ (33% solution in EtOH, 30 ml) and MeOH at a ratio of1:4. The fractions containing the product were evaporated under reducedpressure, redissolved in water and evaporated under reduced pressure.This procedure was repeated 2 to 3 times to afford Compound 6 in freeamine form (90 mg, yield of 84%). The amine was dissolved in water, thepH was adjusted to 6.5 with H₂SO₄ (0.01 M) and lyophilized to give thesulfate salt of Compound 6 as a white foamy solid.

¹H NMR (500 MHz, D₂O, pH=3.5) data of Compound 6 are summarized in Table14 below.

TABLE 14 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.61 d 3.47-3.50 m 3.95-3.97 m3.57-3.59 m 3.68-3.75 m 3.74-3.76 m 3.80-3.83 m J = 4.0 III 5.10 S 4.09d 4.16-4.19 m 3.94-3.96 m 3.60-3.62 m 3.73-3.76 m J = 4.5 H1 H2eq H2axH3 H4 H5 H6 II 3.21-3.25 M 2.39-2.44 m 1.80 ddd 3.45-3.52 m 3.45-3.52 m3.57 t 3.80 t J₁ = 13.0 J = 9.0 J = 10.0 J₂ = J₃ = 12.5

¹³C NMR (125 MHz, D₂O): δ=□□30.0 (C-2), 50.6, 51.4, 54.5, 62.0 (C-5″),62.5 (C-6′), 69.2, 71.3, 74.0, 75.2, 76.5 (2C), 79.6, 81.8, 84.5, 98.5(C-1′), 108.7 (C-1″).

MALDI TOFMS calculated for C₁₇H₃₃N₄O₁₀ Na ([M+Na]⁺) m/e: 478.2; measuredm/e: 478.4.

Preparation of Compound 7a

Compound 18b (500 mg) was dissolved in THF (5 ml) and added to a mixtureof TFA (1 ml) and water (1 ml). The reaction mixture was stirred at 50°C. for 2 hours. Propagation of the reaction was monitored by TLC(EtOAc/Hexane, 7:3). The reaction mixture was purified by flashchromatography (silica, EtOAc/Hexane) to yield Compound 7a (340 mg,yield of 82%).

¹H NMR (500 MHz, CDCl₃): δ=□□1.45-1.52 (m, 1H, H-2 axial), 2.26-2.33 (m,1H, H-2 equatorial), 3.28-3.53 (m, 5H, H-1, H-5, H-6, H-3 and H-4), 3.62(dd, 1H, J₁=3.5 J₂=10.0 Hz H-2′), 3.69-3.73 (m, 2H, H-5″ and H-5′), 3.85(dd, 1H, J₁=3.0 J₂=13.5 Hz, H-5″), 3.86-3.96 (m, 3H, H-4′ and 2H-6′),3.99 (t, 1H, J₁=9.5 J₂=9.0 Hz, H-3′), 4.52-4.55 (m, 1H, H-4″), 5.29 (d,1H, J=4 Hz, H-1′), 5.55 (s, 1H, H-1″), 5.69 (d, 1H, J=5 Hz, H-2″),5.70-5.73 (m, 1H, H-3″), 7.35 (t, 2H, J=7.5 Hz), 7.44 (t, 2H, J=7.5 Hz),7.54 (t, 1H, J=7.5 Hz), 7.60 (t, 1H, J=7.5 Hz), 7.89 (d, 2H, J=8.0 Hz),8.02 (d, 2H, J=8.0 Hz).

¹³C NMR (125 MHz, CDCl₃): δ=32.1 (C-2), 52.3 (C-5″), 58.9, 59.7, 61.0(C-6′), 62.0, 69.3, 71.5, 72.2, 75.4 (2C), 76.0, 80.3, 83.1, 84.1, 98.8(C-1′), 106.9 (C-1″), 128.3-135.5 (12C), 165.2, 165.4.

MALDI TOFMS calculated for C₃₁H₃₄N₁₂O₁₂ Na ([M+Na]⁺) m/e: 789.2;measured m/e: 789.2.

Preparation of Compound 7

Compound 7 was prepared following the chart presented in FIG. 15,starting from Compound 10 and Compound 14b that were converted intoCompound 12 and Compound 15b respectively, that were coupled to oneanother to afford Compound 18b, which was converted into Compound 7d asdescribed hereinabove.

Compound 7a (300 mg, 0.391 mmol) was dissolved in THF (3 ml) and wasadded to a solution of NaOH (0.1M, 2 ml). The reaction mixture wasstirred at room temperature for 10 minutes, and thereafter PMe₃ (1Msolution in THF, 3.3 ml, 3.3 mmol) was added thereto. Propagation of thereaction was monitored by TLC, using a mixture of CH₂Cl₂/MeOH/H₂O/MeNH₂at a relative ratio of 10:15:6:15 diluted to 33% solution in ethanol aseluent, which indicated completion after 5 hours. The reaction mixturewas purified by flash chromatography on a short column of silica gel.The column was washed with the following solvents: THF (100 ml), CH₂Cl₂(200 ml), EtOH (100 ml), and MeOH (150 ml). The product was eluted witha mixture of MeNH₂ (33% solution in EtOH, 40 ml) and MeOH at a ratio of1:4. The fractions containing the product were evaporated under reducedpressure, redissolved in water and evaporated under reduced pressure.This procedure was repeated 2 to 3 times to afford Compound 7 in freeamine form (134 mg, yield of 75%). The amine was dissolved in water, thepH was adjusted to 6.6 with H₂SO₄ (0.01 M) and lyophilized to give thesulfate salt of Compound 7 as a white foamy solid.

¹H NMR (500 MHz, D₂O, pH=3.5) data of Compound 7 are summarized in Table15 below.

TABLE 15 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.74 d 3.53 dd 4.14-4.19 m3.87-3.94 m 3.56-3.59 m 3.77 dd 3.88-3.94 m J = 4.0 J = 4.0, 11.0 J =5.0, 12.0 III 5.25 s 4.23-4.28 m 4.23-4.28 m 4.14-4.19 m 3.16-3.21 m3.36-3.40 m H1 H2eq H2ax H3 H4 H5 H6 II 3.31-3.37 m 2.52 dt 1.89 ddd3.56-3.62 m 3.57-3.63 m 3.70 t 3.90-3.93 m J = 4.0, 12.5 J₁ = J₂ = J₁ =9.5 J₃ = 12.5 J₂ = 9.0

¹³C NMR (125 MHz, D₂O): δ=□□30.0 (C-2), 44.0 (C-5″), 50.6, 51.5, 54.8,61.9 (C-6′), 70.3, 73.4, 74.1, 73.4, 74.1, 75.4, 76.3, 76.5, 79.4, 80.2,81.5, 81.6, 98.3 (C-1′), 110.5 (C-1″);

MALDI TOFMS calculated for C₁₇H₃₃N₄O₁₀ Na ([M+Na]⁺) m/e: 477.2; measuredm/e: 477.2.

Preparation of Compound 8

Compound 8 was prepared following the chart presented in FIG. 16,starting from Compound 10 which was converted into Compound 19c asdescribed hereinabove.

Compound 19c (90 mg, 0.211 mmol) was dissolved in THF (3 ml) and wasadded to a solution of NaOH (0.1M, 2 ml). The reaction mixture wasstirred at room temperature for 10 minutes, and thereafter PMe₃ (1Msolution in THF, 1.69 ml, 1.69 mmol) was added thereto. Propagation ofthe reaction was monitored by TLC, using a mixture ofCH₂Cl₂/MeOH/H₂O/MeNH₂ at a relative ratio of 10:15:6:15 diluted to 33%solution in ethanol as eluent, which indicated completion after 5 hours.The reaction mixture was purified by flash chromatography on a shortcolumn of silica gel. The column was washed with the following solvents:THF (100 ml), CH₂Cl₂ (200 ml), EtOH (100 ml), and MeOH (150 ml). Theproduct was eluted with a mixture of MeNH₂ (33% solution in EtOH, 30 ml)and MeOH at a ratio of 1:4. The fractions containing the product wereevaporated under reduced pressure, redissolved in water and evaporatedunder reduced pressure. This procedure was repeated 2 to 3 times toafford Compound 8 in free amine form (52.0 mg, yield of 76.5%). Theproduct was dissolved in water, the pH was adjusted to 6.6 with H₂SO₄(0.01 M) and lyophilized to afford the sulfate salt of Compound 8.

¹H NMR (500 MHz, D₂O, pH=3.5) data of 8 are summarized in Table 16below.

TABLE 16 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.37 d 3.46-3.51 m 3.98-4.09 m3.98-4.09 m 3.98-4.09 m 3.73 d 3.73 d J = 2.0 J = 5.0 J = 5.0 H1 H2eqH2ax H3 H4 H5 H6 II 3.20-3.26 m 2.42 dt 1.72 ddd 3.41-3.52 m 3.47 t 3.57t 3.80 t J = 4.0, 12.5 J₁ = J₂ = J = 9.5 J = 9.0 J₁ = 9.5, J₃ = 12.5 J₂= 10.0

¹³C (NMR 125 MHz, D₂O): δ=□□29.8 (C-2), 50.1, 50.9, 51.6, 56.0, 61.2(C-6′), 64.9, 73.8, 74.0, 75.8, 82.5, 96.8 (C-1′).

MALDI TOFMS calculated for C₁₂H₂₆N₄O₆ Na ([M+Na]⁺) m/e: 345.2; measuredm/e: 345.2.

Preparation of Compound 9

Compound 9 was prepared following the chart presented in FIG. 17,starting from Compound 10 which was converted into Compound 20c asdescribed hereinabove.

Compound 20c (155 mg, 0.208 mmol) was dissolved in THF (3 ml) and wasadded to a solution of NaOH (0.1M, 2 ml). The reaction mixture wasstirred at room temperature for 10 minutes, and thereafter PMe₃ (1Msolution in THF, 2.28 ml, 2.28 mmol) was added thereto. Propagation ofthe reaction was monitored by TLC, using a mixture ofCH₂Cl₂/MeOH/H₂O/MeNH₂ at a relative ratio of 10:15:6:15 diluted to 33%solution in ethanol as eluent, which indicated completion after 5 hours.The reaction mixture was purified by flash chromatography on a shortcolumn of silica gel. The column was washed with the following solvents:THF (100 ml), CH₂Cl₂ (200 ml), EtOH (100 ml), and MeOH (150 ml). Theproduct was eluted with a mixture of MeNH₂ (33% solution in EtOH, 30 ml)and MeOH at a ratio of 1:4. The fractions containing the product wereevaporated under reduced pressure, redissolved in water and evaporatedunder reduced pressure. This procedure was repeated 2 to 3 times toafford Compound 9 in free amine form (102 mg, yield of 81.4%). The aminewas dissolved in water, the pH was adjusted to 6.5 with H₂SO₄ (0.01 M)and lyophilized to give the sulfate salt of Compound 9 as a yellow foamysolid.

¹H NMR (500 MHz, D₂O, pH=3.75) data of Compound 9 are summarized inTable 17 below.

TABLE 17 Ring H1 H2 H3 H4 H5 H5′ H6 H6′ I 5.72 d 3.45-3.48 m 4.11 t3.79-3.85 m 3.53-3.57 m 3.67-3.69 m 3.80-3.85 m J = 3.5 J = 10.0 H1 H2eqH2ax H3 H4 H5 H6 II 3.24-3.26 m 2.33 dt 1.81 ddd 3.50-3.53 m 3.50-3.53 m3.65 t 3.84 t J = 4.0, 13.0 J₁ = J₂ = 13.0 J = 9.5 J = 10.0 J₃ = 12.5

Additional ¹H NMR (500 MHz, D₂O, pH=3.75) data for Compound 9 included:δ=5.12 (s, 1H).

¹³C (NMR 125 MHz, D₂O): δ=30.0 (C-2″), 50.5, 51.5, 62.0 (C-6′), 70.6,74.2, 75.1, 76.6, 79.1, 80.9, 98.1 (C-1′), 99.6 (C-1″ tether carbon).

MALDI TOFMS calculated for C₂₅H₅₀N₆O₁₄ Na ([M+Na]⁺) m/e: 681.3; measuredm/e: 681.6.

Compound 63 and Compound 10-AHB were prepared from paromamine, viaCompound 62 as illustrated in Scheme 9 below.

Preparation of Compound 62

Zn(OAc)₂ (14.75 grams, 66 mmol) was added to a stirred solution ofparomamine (Compound 1, 9.69 grams, 30 mmol)) in its free base form inH₂O (30 ml) and DMF (150 ml), and the mixture was stirred for 12 hoursat room temperature. A solution of di-tert-butyldicarbonate (9.81 grams,45 mmol) in DMF (20 ml) was added to the reaction mixture over a timeperiod of 30 minutes and the mixture was stirred for an additional 24hours. The reaction progress was monitored by TLC(CH₂Cl₂/MeOH/H₂O/MeNH₂, 10:15:6:15, 33% solution in EtOH). The reactionmixture was diluted with MeOH (250 ml) and loaded onto 50×300 mmion-exchange column (Amberlite, CG50, H⁺ form). The column was washedextensively with 10 column volumes of MeOH/H₂O (60:40) followed byelution with the mixture of MeOH/H₂O/NH₄OH (80:15:5) to yield thedesired N-1-Boc derivative of paromamine at a yield of 40% (5.03 grams).

¹H NMR (500 MHz, D₂O): “Ring I”: δ=2.70 (dd, 1H, J₁=3.5, J₂=7.0 Hz,H-2), 3.26 (dd, 1H, J₁=J₂=9.5 Hz, H-4), 3.43 (dd, 1H, J₁=J₂=9.5 Hz,H-3), 3.62 (dd, 1H, J₁=4.0, J₂=12.5 Hz, H-6′), 3.69 (dt, 1H, J₁=2.0,J₂=10.0 Hz, H-5), 3.72 (dd, 1H, J₁=4.0, J₂=12.5 Hz, H-6), 5.14 (d, 1H,J=4.0 Hz, H-1); “Ring II”: δ=1.18 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H-2ax),2.51 (dt, 1H, J₁=4.5, J₂=12.5 Hz, H-2 eq), 2.73-2.78 (m, 1H, H-3), 3.15(dd, 1H, J₁=J₂=9.5 Hz, H-4), 3.16 (dd, 1H, J₁=J₂=10.0 Hz, H-6),3.29-3.37 (m, 1H, H-1), 3.58 (dd, 1H, J₁=J₂=10.0 Hz, H-5); theadditional peaks in the spectrum were identified as follows: δ=1.30 (s,9H, Boc).

¹³C NMR (125 MHz, D₂O) “Ring I”: δ=56.9 (C-2), 62.4 (C-6), 71.6 (C-4),74.7 (C-5), 77.7 (C-3), 102.6 (C-1); “Ring II”: δ=36.3 (C-2), 50.9(C-3), 52.0 (C-1), 75.3 (C-5), 76.4 (C-6), 88.6 (C-4); the additionalpeaks in the spectrum were identified as follows: δ=29.4 (Boc, 3C),159.6 (Boc, CO).

MALDI TOFMS calculated for C₁₇H₃₃N₃O₉Na ([M+Na]⁺ m/e 446.2; measured m/e446.5).

Thereafter, the two amino groups in product of the previous procedure(44.3 grams, 0.1 mol) were converted to the corresponding azides byfollowing a published procedure [85], using Tf₂O (110 ml, 0.66 mol) andNaN₃ (100 grams, 1.53 mol). The reaction progress was monitored by TLCusing EtOAc 95% and MeOH 5%, which indicated completion after 8 hours.

The crude product was purified by flash chromatography using silica geland an eluent gradient of EtOAc 100% to EtOAc/MeOH (95:5) to yield thecorresponding diazido Compound 62 at a yield of 90% (42.5 grams).

¹H NMR (500 MHz, MeOD): “Ring I”: δ=2.88-2.93 (m, 1H, H-2), 3.23 (dd,1H, J₁=J₂=9.0 Hz, H-4), 3.49 (d, 2H, J₁=3.5 Hz, H-6, H-6′), 3.59-3.63(m, 2H, H-3, H-5), 5.54 (d, 1H, J=3.5 Hz, H-1); “Ring II”: δ=1.08-1.16(m, 1H, H-2ax), 1.96 (dt, 1H, J₁=4.0, J₂=12.5 Hz, H-2 eq), 2.88-2.93 (m,1H, H-6), 3.09-3.16 (m, 1H, H-1), 3.12-3.18 (m, 1H, H-3), 3.18 (dd, 1H,J₁=J₂=9.5 Hz, H-4), 3.23 (dd, 1H, J₁=J₂=9.0 Hz, H-5); the additionalpeaks in the spectrum were identified as follows: δ=□□1.13 (s, 9H, Boc).

¹³C NMR (125 MHz, MeOD) “Ring I”: δ=63.8 (C-6), 66.5 (C-2), 73.1 (C-4),74.5 (C-3), 75.6 (C-5), 101.2 (C-1); “Ring II”: δ=36.2 (C-2), 53.5(C-1), 62.9 (C-3), 77.6 (C-6), 79.1 (C-5), 83.1 (C-4); the additionalpeaks in the spectrum were identified as follows: δ=□□31.1 (Boc, 3C),160.0 (Boc, CO).

MALDI TOFMS calculated for C₁₇H₂₉N₇O₉ K ([M+K]⁺) m/e 514.2; measured m/e514.4).

(S)-2-hydroxy-4-azidobutyric acid, or (S)-4-azido-2-hydroxybutanoicacid, was prepared by the azidation of neomycin following a publishedprocedure [85], using (S)-2-hydroxy-4-aminobutyric acid (80 grams, 0.67mol), Tf₂O (200 ml, 1.20 mol), and NaN₃ (200 grams, 3.00 mol). Thereaction progress was monitored by TLC using EtOAc/MeOH (95:5), whichindicated completion after 8 hours. The reaction mixture wasconcentrated, diluted with EtOAc (2.0 L) and extracted with aqueous HCl(2%) and brine. The combined organic layer was dried over MgSO₄ andconcentrated to yield (S)-2-hydroxy-4-azidobutyric acid at a yield of87% (85 grams).

¹H NMR (300 MHz, MeOD) δ=1.67-1.77 (m, 1H), 1.85-1.93 (m, 1H), 3.29 (t,J=7.0 Hz, 2H), 4.05-4.09 (m, 1H).

¹³C NMR (125 MHz, CDCl₃) δ=36.5, 50.8, 70.8, 179.8.

CIMS calculated for C₄H₇N₃O₃ ⁻ NH₄ ⁺ ([M+K]⁺ m/e 163.1; measured m/e163.1).

Preparation of Compound 63

Compound 62 (3.00 grams, 6.31 mmol) was dissolved in a mixture oftrifluoroacetic acid (12 ml) and dichloromethane (30 ml). The reactionprogress was monitored by TLC using CH₂Cl₂/MeOH (80:20), which indicatedcompletion after 1 hour. The reaction mixture was concentrated todryness under reduced pressure, and the resulting crude was dissolved ina mixture of Et₃N (10 ml) and DMF (10 ml) and cooled to −20° C. In aseparate flask, (S)-2-hydroxy-4-azidobutyric acid (3.94 grams, 31.55mmol) was dissolved in anhydrous DMF (30 ml), cooled to 0° C., and tothe cold solution DCC (7.10 grams, 34.46 mmol) and HOBt (4.72 grams,34.96 mmol) were added. The resulted mixture was stirred at 0° C. forabout one hour. Thereafter, this mixture was carefully added by syringeto the cooled solution of the amine at −20° C. The reaction was stirredat −20° C. for 1 hour and thereafter allowed to warm to room temperaturefor additional 1 hour. Thereafter, the mixture was treated with asolution of MeNH₂ (33% solution in EtOH, 30 ml) and the reactionprogress was monitored by TLC using CH₂Cl₂/MeOH (70:30). Aftercompletion of the reaction (about 8 hours) the mixture was concentratedand purified by flash chromatography (MeOH/CH₂Cl₂) to yield Compound 63at a yield of 93% (3.00 grams).

¹H NMR (500 MHz, MeOD): “Ring I”: δ=3.12 (dd, 1H, J₁=4.0, J₂=10.0 Hz,H-2), 3.45 (dd, 1H, J₁=9.5, J₂=10.5 Hz, H-4), 3.76-3.83 (m, 2H, H-6,H-6′), 3.92 (dd, 1H, J₁=9.0, J₂=10.5 Hz, H-3), 3.97-4.00 (m, 1H, H-5),5.65 (d, 1H, J=3.5 Hz, H-1); “Ring II”: δ=1.55 (ddd, 1H, J₁=J₂=J₃=12.5Hz, H-2ax), 2.18 (dt, 1H, J₁=4.0, J₂=13.0 Hz, H-2 eq), 3.36 (dd, 1H,J₁=J₂=9.0 Hz, H-6), 3.42-3.48 (m, 1H, H-3), 3.50 (dd, 1H, J=J₂=9.0 Hz,H-4), 3.54 (dd, 1H, J₁=J₂=9.0 Hz, H-5), 3.77-3.83 (m, 1H, H-1); theadditional peaks in the spectrum were identified as follows:δ=□□1.82-1.88 (m, 1H, H-9), 2.01-2.07 (m, 1H, H-9), 3.46 (t, 2H, J₁=6.5J₂=7.5, H-10), 4.16 (dd, 1H, J₁=4.0, J₂=8.5, H-8).

¹³C NMR (125 MHz, MeOD) “Ring I”: δ=61.0 (C-6), 63.6 (C-2), 70.6 (C-4),71.3 (C-3), 72.8 (C-5), 98.3 (C-1); “Ring II”: δ=32.3 (C-2), 49.1 (C-1),60.2 (C-3), 74.6 (C-6), 77.4 (C-5), 79.7 (C-4); the additional peaks inthe spectrum were identified as follows: D 8=33.6 (C9), 47.4 (C10), 69.0(C8), 175.7 (C7).

MALDI TOFMS calculated for C₁₆H₂₆N₁₀O₉ K ([M+K]⁺) m/e 541.2; measuredm/e 541.1).

Preparation of Compound 10-AHB

Compound 63 (63 mg, 0.125 mmol) was dissolved in a mixture of THF (1.0ml) and aqueous NaOH (1 mM, 1.5 ml), and was stirred at room temperaturefor 10 minutes. Thereafter PMe₃ (1 M solution in THF, 1.10 ml, 1.10mmol) was added, and the reaction progress was monitored by TLC usingCH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution in EtOH, 10:15:6:15), whichindicated completion after 1 hour. The reaction mixture was purified byflash chromatography on a short column of silica gel. The column waswashed THF (100 ml), CH₂Cl₂ (100 ml), EtOH (50 ml), and MeOH (100 ml).The product was eluted with the mixture of 20% MeNH₂ solution (33%solution in EtOH) in 80% MeOH. Fractions containing the product werecombined and evaporated under reduced pressure. The residue wasdissolved in a small volume of water and evaporated again (2-3 repeats)to afford the free amine form of Compound 10-AHB.

The analytically pure compound was obtained by further chromatography onthe Amberlite CG50 (H⁺ form) column. The column was first washed byMeOH/H₂O 3:2, and then the product was eluted using MeOH/H₂O/NH₄OH(80:10:10) at a yield of 62% (33 mg).

¹H NMR (500 MHz, D₂O, pH=6.5): “Ring I”: δ=3.25 (dd, 1H, J₁=4.0, J₂=11.0Hz, H-2), 3.45 (dd, 1H, J₁=10.0, J₂=10.0 Hz, H-4), 3.64-3.66 (m, 1H,H-6), 3.76-3.82 (m, 1H, H-6′), 3.78 (dd, 1H, J₁=9.0, J₂=10.5 Hz, H-3),3.80-3.85 (m, 1H, H-5), 5.50 (d, 1H, J=3.5 Hz, H-1); “Ring II”: δ=1.60(ddd, 1H, J₁=J₂=J₃=12.5 Hz, H-2ax), 2.18 (dt, 1H, J₁=4.0, J₂=12.5 Hz,H-2 eq), 3.30-3.35 (m, 1H, H-3), 3.44 (dd, 1H, J₁=J₂=9.5 Hz, H-6), 3.54(dd, 1H, J₁=J₂=9.0 Hz, H-5), 3.67 (dd, 1H, J₁=J₂=9.0 Hz, H-4), 3.76-3.82(m, 1H, H-1); the additional peaks in the spectrum were identified asfollows: δ=1.88-1.92 (m, 1H, H-9), 2.02-2.08 (m, 1H, H-9), 2.99-3.07 (m,2H, H-10), 4.21 (dd, 1H, J₁=4.0, J₂=8.0, H-8).

¹³C NMR (125 MHz, D₂O) “Ring I”: δ=56.0 (C-2), 62.2 (C-6), 71.3 (C-4),71.5 (C-5), 75.2 (C-3), 99.2 (C-1); “Ring II”: δ=32.6 (C-2), 50.4 (C-1),51.1 (C-3), 75.3 (C-6), 77.2 (C-5), 83.6 (C-4); the additional peaks inthe spectrum were identified as follows: □δ=□ 32.5 (C9), 38.3 (C10),71.3 (C8), 177.3 (C7).

MALDI TOFMS calculated for C₁₆H₃₂N₄O₉ K ([M+K]⁺) m/e 463.2; measured m/e463.1.

Compound 64, Compound 65 and Compound 37 (also referred to herein asNB54) were prepared as illustrated in Scheme 10 below.

Preparation of Compound 64:

Compound 63 (3.00 grams, 5.85 mmol) was dissolved in dry pyridine (10ml), cooled at −12° C. and then acetic anhydride (5.2 equivalents, 3.00ml) was added. The reaction temperature was kept at −12° C. and thereaction progress was monitored by TLC using EtOAc/Hexane (70:30), whichindicated completion after 8 hours. The reaction mixture was dilutedwith EtOAc and extracted with HCl (2%), saturated aqueous NaHCO₃, andbrine. The combined organic layer was dried over MgSO₄ and concentrated.The crude product was purified by flash chromatography using silica geland EtOAc/Hexane as eluent to afford Compound 64 at a yield of 75% (3.15grams).

¹H NMR (500 MHz, CDCl₃): “Ring I”: δ=3.70 (dd, 1H, J₁=3.0, J₂=11.0 Hz,H-2), 4.10 (dd, 1H, J₁=2.0, J₂=12.5 Hz, H-6), 4.31 (dd, 1H, J₁=4.5,J₂=12.5 Hz, H-6′), 4.36-4.39 (m, 1H, H-5), 5.05 (dd, 1H, J₁=10.0,J₂=10.0 Hz, H-4), 5.28 (d, 1H, J=3.5 Hz, H-1) 5.50 (dd, 1H, J₁=10.0,J₂=10.0 Hz, H-3); “Ring II”: δ=1.48 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H-2ax),2.50 (dt, 1H, J₁=4.0, J₂=13.0 Hz, H-2 eq), 3.34-3.39 (m, 1H, H-4),3.38-3.43 (m, 1H, H-3), 3.74-3.78 (m, 1H, H-5), 3.99-4.04 (m, 1H, H-1),4.82 (dd, 1H, J₁=J₂=10.0 Hz, H-6), 6.63 (d, 1H, J=7.5 Hz, NH); theadditional peaks in the spectrum were identified as follows: δ=□2.05-2.09 (m, 2H, H-9), 2.05 (s, 3H, Ac), 2.08 (s, 3H, Ac), 2.09 (s, 3H,Ac), 2.15 (s, 3H, Ac), 2.19 (s, 3H, Ac), 3.35-3.38 (m, 2H, H-10), 5.14(dd, 1H, J₁=5.5, J₂=6.5, H-8).

¹³C NMR (125 MHz, CDCl₃) “Ring I”: δ=61.8 (C-6), 61.8 (C-2), 68.1 (C-4),68.4 (C-5), 71.4 (C-3), 99.1 (C-1); “Ring II”: δ=32.5 (C-2), 48.2 (C-1),58.2 (C-3), 73.8 (C-5), 74.0 (C-6), 84.3 (C-4); the additional peaks inthe spectrum were identified as follows: □δ=□20.6-20.9 (Ac, 5C), 30.5(C-9), 47.1 (C-10), 70.8 (C8), 169.1 (C-8, CO), 169.8 (Ac, CO), 169.8(C-7, CO), 170.0 (Ac, CO), 170.6 (Ac, CO), 172.4 (Ac, CO).

MALDI TOFMS calculated for C₂₆H₃₆N₁₀O₁₄ K ([M+K]⁺) m/e 751.1; measuredm/e 752.2).

Preparation of Compound 65

Anhydrous CH₂Cl₂ (10 ml) was added to powdered, flame-dried 4 Åmolecular sieves (3.00 grams), followed by the addition of the acceptorCompound 64 (1.75 grams, 2.46 mmol) and the donor5-deoxy-5-azido-2,3-di-O-benzoyl-1-O-tricloroacetymido-D-ribofuranose(3.30 grams, 6.27 mmol) dissolved in CH₃CN (10 ml). The mixture wasstirred for 10 minutes at room temperature and was then cooled down to−20° C. A catalytic amount of BF₃-Et₂O (100 μl) was added to thereaction mixture, and the mixture was stirred at −15° C. The reactionprogress was monitored by TLC using EtOAc/Hexane (60:40), whichindicated completion after 30 minutes. The reaction was diluted withCH₂Cl₂ and filtered through celite. After thorough washing of celitewith CH₂Cl₂, the washes were combined and extracted with saturatedaqueous NaHCO₃, brine, dried over MgSO₄ and concentrated. The crudeproduct was purified by flash chromatography to afford Compound 65 at ayield of 76% (2.01 grams) and Compound 64 at a yield of 20% (350 mg).

¹H NMR (500 MHz, CDCl₃): “Ring I”: δ=3.53 (dd, 1H, J₁=4.0, J₂=11.0 Hz,H-2), 4.15 (dd, 1H, J₁=2.0, J₂=12.5 Hz, H-6), 4.26 (dd, 1H, J₁=4.0,J₂=12.5 Hz, H-6′), 4.50-4.55 (m, 1H, H-5), 5.07 (dd, 1H, J₁=9.5, J₂=10.0Hz, H-4), 5.43 (dd, 1H, J₁=9.5, J₂=10.0 Hz, H-3), 5.83 (d, 1H, J=4.0 Hz,H-1); “Ring II”: δ=1.47 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H-2ax), 2.50 (dt,1H, J₁=4.0, J₂=13.0 Hz, H-2 eq), 3.56-3.58 (m, 1H, H-3), 3.72 (dd, 1H,J₁=8.5, J₂=9.5 Hz, H-4), 3.99 (dd, 1H, J₁=8.5, J₂=9.5 Hz, H-5),4.01-4.08 (m, 1H, H-1), 4.91 (dd, 1H, J₁=9.5, J₂=10.5 Hz, H-6), 6.64 (d,1H, J=8.5 Hz, NH); “Ring II” I: δ=3.57 (dd, 1H, J₁=6.0, J₂=13.0 Hz,H-5′), 3.64 (dd, 1H, J₁=3.0, J₂=13.0 Hz, H-5), 4.50-4.55 (m, 1H, H-4),5.49 (dd, 1H, J₁=5.0, J₂=7.0 Hz, H-3), 5.62 (dd, 1H, J₁=1.0, J₂=5.0 Hz,H-2), 5.68 (d, 1H, J=1.0 Hz, H-1); the additional peaks in the spectrumwere identified as follows: δ=□□2.00-2.15 (m, 2H, H-9), 2.04 (s, 3H,Ac), 2.09 (s, 6H, 2Ac), 2.20 (s, 3H, Ac), 2.23 (s, 3H, Ac), 3.35 (t, 2H,J=7.0, H-10), 5.16 (dd, 1H, J₁=5.0, J₂=7.0, H-8), 7.33 (t, 2H, J=8.0,Bz), 7.43 (t, 2H, J=8.0, Bz), 7.51-7.54 (m, 1H, Bz), 7.56-7.60 (m, 1H,Bz), 7.85 (d, 2H, J=1, 7.5, Bz), 7.94 (dd, 2H, J=1, 7.5, Bz).

¹³C NMR (125 MHz, CDCl₃) “Ring I”: δ=61.6 (C-2), 61.8 (C-6), 68.1 (C-5),68.2 (C-4), 70.7 (C-3), 96.8 (C-1); “Ring II”: δ=32.1 (C-2), 48.4 (C-1),58.5 (C-3), 73.5 (C-6), 77.9 (C-4), 80.0 (C-5); “Ring II” I: δ=52.7(C-2), 71.5 (C-3), 74.7 (C-2), 80.1 (C-4), 107.4 (C-1); the additionalpeaks in the spectrum were identified as follows: □δ=□20.6-20.9 (Ac,5C), 30.5 (C-9), 47.1 (C-10), 70.9 (C8), 128.4 (Bz, 2C), 128.5 (Bz, 2C),129.6 (Bz, 2C), 129.7 (Bz, 2C), 133.6 (Bz, 1C), 133.7 (Bz, 1C), 165.2(Bz, CO), 165.2 (Bz, CO), 168.9 (C-8, CO), 169.7 (Ac, CO), 169.7 (C-7,CO), 169.9 (Ac, CO), 170.6 (Ac, CO), 172.3 (Ac, CO).

MALDI TOFMS calculated for C₄₅H₅₁N₁₃O₁₉ K ([M+K]⁺) m/e 1116.3; measuredm/e 1116.3.

Preparation of Compound 37

Compound 65 (1.55 grams, 1.44 mmol) was treated with a solution of MeNH₂(33% solution in EtOH, 50 ml) and the reaction progress was monitored byTLC using EtOAc/MeOH (85:15), which indicated completion after 8 hours.The reaction mixture was evaporated to dryness and the residue wasdissolved in a mixture of THF (5 ml) and aqueous NaOH (1 mM, 5.0 ml).This mixture was stirred at room temperature for 10 minutes, after whichPMe₃ (1 M solution in THF, 11.52 ml, 11.52 mmol) was added. The reactionprogress was monitored by TLC using CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solutionin EtOH) (10:15:6:15), which indicated completion after 1 hour.

The reaction mixture was purified by flash chromatography on a shortcolumn of silica gel. The column was washed with the following solvents:THF (800 ml), CH₂Cl₂ (800 ml), EtOH (200 ml), and MeOH (400 ml). Theproduct was then eluted with the mixture of 20% MeNH₂ solution (33%solution in EtOH) in 80% MeOH. Fractions containing the product werecombined and evaporated under reduced pressure. The residue wasre-dissolved in a small volume of water and evaporated again (2-3repeats) to afford the free amine form of Compound 37 (also referred toas NB54).

The analytically pure product was obtained by eluting the above productthrough a short column of Amberlite CG50 (NH₄ ⁺ form). The column wasfirst washed with MeOH/H₂O (3:2), then the product was eluted with amixture of MeOH/H₂O/NH₄OH (80:10:10) to afford Compound 37 at a yield of78% (628 mg).

The product was dissolved in water, the pH was adjusted to 6.5 by H₂SO₄(0.1 N) and lyophilized to afford the sulfate addition salt of Compound37, which was used in all biological studies.

¹H NMR (500 MHz, D₂O, pH=3.5): “Ring I”: δ=3.52 (dd, 1H, J₁=4.0, J₂=11.0Hz, H-2), 3.53-3.57 (m, 1H, H-4), 3.81-3.85 (m, 1H, H-5), 3.81-3.85 (m,1H, H-6), 3.93-3.98 (m, 1H, H-6′), 4.04 (dd, 1H, J₁=9.0, J₂=10.0 Hz,H-3), 5.87 (d, 1H, J=4.0 Hz, H-1); “Ring II”: δ=1.80 (ddd, 1H,J₁=J₂=J₃=12.5 Hz, H-2ax), 2.24 (dt, 1H, J₁=4.0, J₂=12.5 Hz, H-2 eq),3.54-3.58 (m, 1H, H-3), 3.73 (dd, 1H, J₁=J₂=9.5 Hz, H-6), 3.92-3.98 (m,1H, H-1), 3.99 (dd, 1H, J₁=J₂=9.0 Hz, H-5), 4.11 (dd, 1H, J=J₂=9.5 Hz,H-4); “Ring III”: δ=3.29 (dd, 1H, J₁=7.0, J₂=13.5 Hz, H-5), 3.42 (dd,1H, J₁=4.0, J₂=13.5 Hz, H-5′), 4.12-4.15 (m, 1H, H-4), 4.23-4.25 (m, 1H,H-3), 4.26-4.29 (m, 1H, H-2), 5.40 (s, 1H, H-1); the additional peaks inthe spectrum were identified as follows: δ=□□1.98-2.06 (m, 1H, H-9),2.15-2.22 (m, 1H, H-9), 3.14-3.23 (m, 2H, H-10), 4.34 (dd, 1H, J₁=4.0,J₂=8.5, H-8).

¹³C NMR (125 MHz, D₂O) “Ring I”: δ=55.4 (C-2), 61.9 (C-6), 70.8 (C-3),70.8 (C-4), 75.8 (C-5), 95.7 (C-1); “Ring II”: δ=31.4 (C-2), 50.4 (C-3),51.4 (C-1), 74.6 (C-6), 78.1 (C-4), 85.1 (C-5); “Ring III”: δ=43.4(C-5), 72.7 (C-3), 76.5 (C-3), 79.9 (C-4), 110.5 (C-1); the additionalpeaks in the spectrum were identified as follows: Dδ=D 32.6 (C9), 38.4(C10), 71.3 (C8), 177.4 (C7).

MALDI TOFMS calculated for C₂₁H₄₁N₅O₁₂ K ([M+K]⁺) m/e 594.3; measuredm/e 594.3.

Example 2 Biological Activity Assays

The ability of the compounds presented herein, i.e., Compounds 2-9, toread-through stop codon mutations was examined both in-vitro and ex-vivoin mammalian cultured cells.

All chemicals and reagents were obtained from common commercial sourcesunless otherwise stated. The commercial antibiotics, paromomycin andgentamicin were obtained from Sigma.

In Vitro Translation Reactions and Quantification of Suppression andTranslation:

The mutation suppression activity assays were performed on “UGA C”mutation, which was shown to be most susceptible foraminoglycoside-mediated suppression. Initially, Compounds 1-9 weretested for their ability to suppress this nonsense mutation in vitro,using a reporter construct carrying the R3X nonsense mutation (apremature UGA C stop codon) of the PCDH15 gene. Mutations in the PCDH15gene which encodes protocardherin 15, cause type 1 Usher syndrome(USH1), which is characterized by profound prelingual hearing loss,vestibular areflexia, and prepubertal onset of retinitis pigmentosa (RP)[87]. Four different PCDH15 USH1-causing nonsense mutations, R3X, R245X,R643X, and R929X, have been reported in humans. Interestingly, while theabove nonsense mutations of PCDH15 cause USH1, certain missensemutations in the same gene cause only nonsyndromic deafness, which isnot associated with RP. Such observations suggest that partial or lowlevel activity of the protein encoded by this gene may be sufficient fornormal retinal function, making any of the compounds presented herein asuitable candidate for read-through therapy.

Suppression of nonsense mutations by Compounds 1-9 was tested in vitrousing a reporter plasmid harboring the R3X mutation of the PCDH15 gene[88]. To create this plasmid, the oligonucleotides5′-GATCCATGTTTTGACAGTTTTATCTCTGGACA-3′ (SEQ ID NO: 1) and5′-AGCTTGTCCAGAGATAAAACTGTCAAAACATG-3′ (SEQ ID NO: 2) were annealed toeach other and inserted into the BamHI and HindIII sites of plasmidpDB650 [6].

The resulting reporter plasmid pDB650-R3X contained a TGA C nonsensemutation between a 25-kDa polypeptide encoding open reading frame (ORF)and a 10-kDa polypeptide encoding ORF. Hence, an efficient translationtermination at the stop codon resulted in the production of a 25-kDapolypeptide, while suppression of the nonsense mutation by the compoundstested herein allowed the synthesis of a longer 35-kDa protein.

The plasmid was transcribed and translated in a rabbit reticulocytelysate coupled transcription/translation system (Promega) in thepresence of [³⁵S]-methionine, and the reaction products were separatedby SDS-PAGE and quantified using PhosphorImager analysis. The mutationsuppression level was calculated as the relative proportion of the35-kDa product out of total protein (the sum of 35-kDa and 25-kDa), andthe translation level was calculated as the relative proportion of thetotal protein at each tested compound concentration out of the totalprotein without the presence of the tested compounds.

The mutation suppression activities of paromomycin and gentamicin weremeasured and used as reference to the activity of the compoundspresented herein. The tested concentrations of paromomycin were 0, 5,10, 20, 30 and 40 μg/ml, tested concentrations of gentamicin were 0, 5,10, 20, and 30 μg/ml, and the tested concentrations of Compounds 1-9were in the range of 0-160 μg/ml. The concentrations at which maximalsuppression levels were observed are given in Table 18 below.

Using similar methods for testing and analyses, compounds which exhibitan N1-AHB group as presented hereinabove are tested for mutationsuppression activity.

Table 18 presents maximal in-vitro mutation suppression and translationlevels of the R3X mutation, along with the MIC values measured forCompounds 1-9. The results in Table 18 are reported as averages of atleast three independent experiments.

TABLE 18 MIC [μg ml⁻¹] Conc. Supp. Trans. B. Compound [μg ml⁻¹] level(%) level (%) E. coli Subtilis Paromomycin 40  49 ± 6  40 ± 13 12 8Gentamicin 30  49 ± 4 40 ± 9 4 <0.5 Paromamine 80   6.2 ± 0.2  74 ± 15512 128 (Compound 1) Compound 2 80   1.3 ± 0.1 100 ± 10 256 64 Compound3 80  21 ± 3 72 ± 6 >512 48 Compound 4 80   1.5 ± 0.1 74 ± 7 256 96Compound 5 80 4.4 ± 2 75 ± 9 >512 192 Compound 6 160 <1 82 ± 8 >512 >512Compound 7 80 2.9 ± 2  98 ± 10 192 48 Compound 8 80 2.0 ± 2 71 ± 7 19248 Compound 9 80 <1 22 ± 2 96 48

As can be seen in Table 18, removal of either one ring which is presentin paromomycin, namely ring IV as in the case of Compound 2, or tworings which are present in paromomycin, namely rings III and IV as inthe case of Compound 1, dramatically decreases its in vitro read-throughactivity from 49% suppression to 1.6% (Compound 2) and 6.2% (Compound1). These data alone indicate that ring IV of paromomycin is criticalfor its proper recognition of the mammalian A-site and for itssubsequent read-through activity. The substantially higher suppressionlevel of Compound 1 (6.2%) compared to that of the Compound 2 (1.6%)implies that Compound 1 (paromamine) represents the minimal structuralmotif of paromomycin that is preferentially recognized by the mammalianribosome and has significant suppression activity.

As can further be concluded from Table 18, connection of the plainribose ring to the paromamine scaffold either at the C6 position, as inCompound 4, or at the C3′ position as in Compound 6, along with theaddition of one extra amine as in Compound 8, or paromamine dimerizationas in Compound 9, gave lower suppression levels than that of paromamineitself.

The most important results, however, were observed when instead of plainribose the 5-amino ribose (ribosamine) was connected to the paromaminemoiety at different positions. As can be also seen in Table 18, theobserved mutation suppression levels of Compounds 3, 5, and 7 werehigher than the corresponding compounds containing plain ribose ring atthe same position, namely Compounds 2, 4, and 6 respectively. Inaddition, in the series of Compounds 3, 5, and 7, a particular influenceof the position of the ribosamine on the paromamine scaffold wasobserved to be C5 (Compound 3)>>C6 (Compound 5)>C3′ (Compound 7),suggesting that the preservation of the pseudo-trisaccharide corestructure of the parent paromomycin (rings I-III) in Compound 3 isimportant for efficient read-through activity.

The mutation suppression data of Compounds 1-9 show that, although inthe series of Compounds 2-7, an increased number of amino groups in eachpair leads to improved read-through activity, the data obtained withCompound 8 and Compound 9 indicate that merely increasing the number ofamino groups on the paromamine scaffold does not always lead to anincrease in read-through activity, even though the binding affinity ofthese analogs to both prokaryotic and eukaryotic rRNA is likely to beincreased [36, 89]. Nevertheless, the observed 13-fold highersuppression level of Compound 3 compared to that of the correspondingribose Compound 2, and over 3-fold higher activity compared to that ofCompound 1, suggest that the presence of C5″-NH₂ group in Compound 3 isresponsible for its elevated read-through activity.

FIGS. 3a-d present the results of the in vitro mutation suppression andtranslation assays measured for the exemplary Compound 3, andparomomycin, by expression of a plasmid-based reporter constructcontaining a TGA C nonsense stop mutation between a 25-kDa polypeptideencoding open reading frame (ORF) and a 10-kDa polypeptide encoding ORF,in the presence of the tested compounds and [³⁵S]-methionine, showingthe reaction products separated by SDS-PAGE and quantified using aphosphor-imager for Compound 3 (FIG. 3a ) and paromomycin (FIG. 3c ),and showing comparative plots where the mutation suppression values(shown in black dots) and the translation values (shown in white dots),calculated as the relative proportion of the total protein at eachconcentration of the tested compounds out of the total protein expressedin the absence thereof, as measured in triplicates for Compound 3 (FIG.3b ) and for paromomycin (FIG. 3d ).

As can be seen in Table 18 and FIGS. 3a-d , besides its very significantread-through activity, Compound 3 also retained about two-fold highertranslation level (about 80%, see also Table 18) than either paromomycin(about 40%) or gentamicin (about 40%), at the concentrations in whicheach tested compound reached maximal suppression rate. Such a reductionin translation inhibition by Compound 3 could be interpreted as areduced toxicity of Compound 3 relative to that of the parentparomomycin or gentamicin compounds. Thus, although at the aboveconcentrations the paromomycin-induced suppression rate is higher thanthat of Compound 3, the amount of total protein produced in the case ofCompound 3 is larger.

Ex Vivo Mutation Suppression Induced by Aminoglycoside Antibiotics:

While further reducing the present invention to practice, it wasreasoned that the protein production enhancement exhibited by Compound3, even at the observed suppression rate, could in principle increasethe efficacy of drug-induced nonsense suppression in mammalian cellsystems, by increasing the total amount of functional proteins producedfrom nonsense codon-containing genes. Recent studies on enhancedproduction of functional proteins from nonsense codon-containing genesby promoter-activating agents support this presumption [33]. To testthis possibility, Compound 3 along with paromomycin and gentamicin, wereevaluated for the UGA C stop codon read-through activity in culturedcells using a dual luciferase reporter system.

Suppression of nonsense mutations by Compounds 1-9 was tested ex vivousing a dual luciferase reporter plasmid [90]. The p2LUC plasmidharboring the UGAC mutation was transfected to COS-7 cells withLipofectamine 2000 (Invitrogen) and addition of tested compounds wasperformed after 15 hours. Luciferase activity was determined after 24hours of incubation, using the Dual Luciferase Reporter Assay System(Promega) and stop codon readthrough was calculated as describedpreviously.

Using similar methods for testing and analyses, compounds which exhibitan N1-AHB group as presented hereinabove are tested for ex-vivo mutationsuppression activity.

FIG. 4 presents the ex-vivo suppression of a nonsense mutation exhibitedby the exemplary Compound 3, paromomycin and gentamicin, using the p2Lucplasmid containing a TGA C nonsense mutation in a polylinker between therenilla luciferase and firefly luciferase genes expressed in COS-7cells, showing the calculated suppression levels as averages of threeindependent experiments or more for each tested compound at differentconcentrations.

As can be seen in FIG. 4, the activity of Compound 3 is superior to thatof paromomycin and gentamicin at all the tested concentrations. In thecompounds tested, the induced read-through activity increased with theincreased concentration of the tested compound, but this increase wasmore significant in the case of Compound 3 than the other two clinicallyused drugs. As can further be seen in FIG. 4, gentamicin, which iscurrently the only clinically relevant aminoglycoside shown to have theability to suppress nonsense mutations in patients, was less efficientthan either paromomycin or Compound 3. Such an increased read-througheffectiveness of Compound 3 relative to those of paromomycin andgentamicin is most likely caused due to its lower toxicity with respectto the cultured cells.

Antibacterial Activity:

In order to compare the observed aminoglycoside-induced stop codonread-through activity in mammalian cells to antibacterial activitythereof, Compounds 1-9 were further investigated as antibacterial agentsagainst both Gram-negative (E. coli) and Gram-positive (Bacillussubtilis) bacteria, and the minimal inhibitory concentration (MIC)values were determined by using a microdilution assay with paromomycinand gentamicin as controls, and the results are presented in Table 18hereinabove.

The bacterial strains used were Escherichia coli ATCC 25922, andBacillus Subtilis ATCC 6633. The MIC values were determined using thedouble-microdilution method according to the National Committee forClinical Laboratory Standards (NCCLS) [91] with two different startingconcentrations of the tested compounds, 384 μg/ml and 512 μg/ml. All theexperiments were performed in triplicates and analogous results wereobtained in two to four different experiments.

Using similar methods for testing and analyses, compounds which exhibitan N1-AHB group as presented hereinabove are tested for antimicrobialactivity.

As can be deduced from the MIC values presented in Table 18, theantibacterial activity of all the compounds presented herein is markedlylower than that of the parent compound paromomycin by a factor whichranges from 8 to 43 in E. coli and from 6 to 64 in B. subtilis, whereasgentamicin and paromomycin exhibited excellent antibacterial activityagainst both bacterial strains.

Most importantly, the considerably lower antibacterial activity ofCompound 3 compared to that of paromomycin indicates that theselectivity of Compounds 3 to the eukaryotic cells is much higher thanthat of the parent paromomycin. Thus, the observed inability ofCompounds 3 to show significant antibacterial activity should beinterpreted as a positive result in the sense of the generalapplicability of the strategy outlined herein for the design of newvariants of aminoglycosides that can act selectively on the mammalianribosome and cause efficient stop codon suppression without upsettingthe GI biota equilibrium or increasing the emergence of resistance toantibiotics.

Cytotoxicity in Mammalian System:

To further confirm the reduced toxicity of Compound 3 with reference tocommercially available and clinically-used aminoglycosides, a series ofcell toxicity assays was performed using three kidney-derived celllines, i.e., HEK-293 (human embryonic kidney), COS-7 (monkey kidney),and MDCK (canine kidney), as described hereinbelow.

HEK-293, COS-7 or MDCK were grown in 96-well plates (5000 cells/well) inDMEM medium containing 10% FBS, 1% penicillin/streptomycin and 1%glutamine (Biological Industries) at 37° C. and 5% CO₂ over night. After1 day the medium was changed to medium without streptomycin anddifferent concentrations of the tested compounds were added. After 48hours a cell proliferation assay (XTT based colorimetric assay,Biological Industries) was performed, using the 5 hours incubationprotocol, according to the manufacturer's instructions. Optical density(OD) was read using an Elisa plate reader. Cell viability was calculatedas the ratio between the number of living cells in cultures grown in thepresence of the tested compounds, versus the corresponding cell cultureswhich were grown without the tested compounds. The concentration ofhalf-maximal lethal dose for cells (LC₅₀) was obtained from fittingconcentration-response curves to the data of at least two independentexperiments, using Grafit 5 software [R. J. Leatherbarrow, ErithacusSoftware Ltd., Horley, U.K. 2001](see, Table 19 below).

Using similar methods for testing and analyses, compounds which exhibitan N1-AHB group as presented hereinabove are tested for cytotoxicity.

TABLE 19 LC₅₀ values for cell line (mg/ml) Compound HEK293 COS-7 MDCKgentamicin 0.70 ± 0.08 0.49 ± 0.06 0.51 ± 0.07 paromomycin 0.77 ± 0.140.90 ± 0.10 1.00 ± 0.06 Compound 3 4.82 ± 0.92 7.03 ± 1.85 7.53 ± 1.21

As can be seen in Table 19, the LC₅₀ values obtained for Compound 3 were6-fold to 15-fold higher than the LC₅₀ values obtained for theclinically-used aminoglycosides gentamicin and paromomycin as tested inall three cell lines.

In summary, the paromamine derivatives, Compounds 2-9, synthesized andtested by the present inventors, provide a systematic study of syntheticaminoglycosides for suppression of premature stop codons in both invitro and ex vivo mammalian translation systems. Paromamine (Compound 1)was identified as the minimal structural motif of the clinicallyimportant drug paromomycin and was used as a scaffold for theconstruction of diverse structures as potential stop codon read-throughinducers. These compounds showed significantly higher stop codonread-through activity and lower toxicity compared to that of the parentparomomycin in cultured cells. In COS-7 cells the activity of thesecompounds was also higher than that of gentamicin, the onlyaminoglycoside to date that was shown to have the ability to suppressnonsense mutations in patients. Antibacterial tests against bothGram-negative and Gram-positive bacterial strains indicate that thesecompounds are highly selective in their action in eukaryotic cells.

Based on the experimental results presented hereinabove, it can be seenthat the mutation read-through inducing activity of the compoundsaccording to the present embodiments is improved notably as measured exvivo in mammalian cells and compared to the read-through activity ofcommercially availably and clinically used aminoglycosides. Moreover,the reduced toxicity of the compounds presented herein combined withtheir nonsense mutations suppression activity renders the use thereofhighly advantageous as compared to commercially availably and clinicallyused aminoglycosides, since it is expected to be accompanied by fewerand more minor side effects. These observations clearly demonstrate thatthe compounds presented herein can be regarded as a potential newtherapeutic agent for the treatment of genetic disorders caused bytruncation mutations.

Taken together, these results suggest that the compounds presentedherein could represent an alternative to gentamicin and paromomycin formutation suppression therapy, thus providing a new direction for thedevelopment of novel aminoglycoside-based small molecules that targetmammalian cells selectively by means of optimizing the efficiency ofaminoglycoside-induced suppression of premature stop mutations.

Example 3 Activity of Exemplary Compound 37

An exemplary compound of the compounds presented herein, Compound 37,was selected for further comparative studies regarding its capacity toread-through, and thus suppress the expression of stop codon mutationsboth in-vitro and ex-vivo in mammalian cultured cells. The assays wereconducted in comparison to another potent compound according to thepresent embodiments, Compound 3, and to other known aminoglycosides.

All chemicals and reagents were obtained from common commercial sourcesunless otherwise stated.

Comparative In Vitro Suppression of PCDH15 Nonsense Mutations, UnderlingType 1 Usher Syndrome, by Various Aminoglycosides:

Compound 37 was tested for its ability to suppress a nonsense mutationin-vitro, using a reporter construct carrying either R3X nonsensemutation (a premature UGA C stop codon) or R245X nonsense mutation (apremature UGA A stop codon) of the PCDH15 gene, using gentamicin,paromomycin and Compound 3 as standards and following the experimentalprocedures presented hereinabove.

Each compound was assayed at several different concentrations and theconcentrations at which maximal suppression levels were observed aregiven in Table 20.

Table 20 below presents the maximum in vitro suppression levels achievedfor R3X and R245X mutations. The results were obtained at aminoglycosideconcentration for which maximal suppression level was achieved. Thedetermination of the suppression and translation levels were performedas previously reported [6]. Each compound was assayed at severaldifferent concentrations and the concentrations at which maximalsuppression levels were observed are given. The results are averages ofat least three independent experiments.

TABLE 20 R245X R3X Concen- Concentration suppression tration suppressionAminoglycoside (μM)^(a) level (%)^(b) (μM)^(a) level (%)^(b) gentamicin46 50 ± 3 61  11 ± 1.5 paromomycin 56 49 ± 6 42 9 ± 3 Compound 3 142 21± 9 178 6.0 ± 0.3 Compound 37 153 71 ± 9 153 40.5 ± 7.5 

As can be seen in Table 20, Compound 37, in which the AHB moiety isattached at N-1 position, shows the highest suppression levels than thatof its parent Compound 3, as well as than those of paromomycin andgentamicin. The maximal suppression level of Compound 37 (71%) for R3Xnonsense mutation is over three-fold higher than that of Compound 3(21%). Furthermore, it has been previously shown that both the basal andaminoglycoside-induced suppression of UGA A stop codon is much lessefficient than that of the UGA C stop codon [6]. Although the sequencecontest around the stop codon has also significance on the suppressionefficiency [23, 92] and as such it was expected that the suppressionlevels of R3X and R245X would be different, it was expected that sinceR245X consists of the UGA A stop sequence its suppression byaminoglycosides could be less efficient than that of R3X that consistsof a UGA C stop sequence. Indeed, the maximal suppression levels of allaminoglycosides tested, the suppression of R245X nonsense mutation aresignificantly lower that of the R3X nonsense mutation.

As can further be seen in Table 20, while the difference in suppressionof R3X versus R245X in gentamicin, paromomycin and Compound 3, are inthe order of 4.5-, 5.4- and 3.5-fold, respectively, only 1.8-folddifference is observed for Compound 37. Moreover, Compound 37 with amaximal suppression level of 40.5% of the R245X nonsense mutation, is3.6-, 4.5- and 6.5-fold better than gentamicin, paromomycin and Compound3, respectively, and turns out to the best aminoglycoside derivative forthe suppression of this mutation. It is note that although the maximalsuppression level of Compound 37 was obtained at 100 μM, a concentrationwhich is much higher than the concentrations required for the maximalactivities of both gentamicin and paromomycin, the preference ofCompound 37 over gentamicin, paromomycin and Compound 3, was alsoobserved at the same concentrations of Compound 37 and these compounds.

Comparative In Vitro Suppression of PCDH15, CFTR, Dystrophin and IDUANonsense Mutations by Various Aminoglycosides, Using Dual LuciferaseReporter System:

The impact of Compound 37 on other genetic diseases was tested on modelsof the following genetic diseases.

Cystic Fibrosis (CF)—

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloridechannel which controls the regulation of chloride and sodium transportin secretory epithelial cells [93]. Mutations in the CFTR gene have beenfound to cause CF, a recessive hereditary disorder. Different mutationscause defects in CFTR production and function by different molecularmechanisms: production of defective proteins, faulty protein processingor regulation, malfunctioning proteins or mutations affecting the levelsor the processing of mRNA. Nonsense mutations account for approximately10% of the total mutant alleles in CF patients. Among them the G542Xmutation constitutes roughly 2.5% of all nonsense mutations. However, incertain populations the incidence of nonsense mutations is much higher,namely in Ashkenazi Jews the W1282X is the most common mutation andtogether with other nonsense mutations accounts for 64% of all CFalleles [93, 94].

Duchenne Muscular Dystrophy (DMD)—

DMD is a severe X-linked neuromuscular disorder with an incidence of1/3,500 male births, which results from mutations in the gene thatencodes the dystrophin protein [95, 96]. The most distinctive feature ofDMD is a progressive proximal muscular dystrophy with characteristicpseudohypertrophy of the calves. The onset usually occurs before the ageof three years, and the victim is chair-ridden by the age of twelve anddead by the age of twenty. Approximately two-thirds of the mutations inboth forms are deletions of one or many exons in the dystrophin gene.The vast majority of DMD point mutations result in premature translationtermination, namely are nonsense mutations; these account forapproximately 5-13% of the muscular dystrophies. It seems likely thatmost cases of DMD arise as a result of a reduction in the level ofdystrophin transcripts [97].

Usher Syndrome (USH)—

USH is estimated to be the most frequent cause of deaf-blindness in theUnited States. It is characterized by congenital profound sensorineuralhearing loss, vestibular areflexia, and retinitis pigmentosa, with onsetnear puberty [98]. The frequency of USH was estimated to be from1/16,000 to 1/50,000 in various populations [99]. It was determined thatthe carrier frequencies of the nonsense mutation R245X are 0.79% and2.48% among Ashkenazi Jews from New York and Israel, respectively, thusbeing the major cause of USH Type IF in Ashkenazi Jewish population[100].

Hurler Syndrome—

Hurler Syndrome or Mucopolysaccharidosis type IH (MPSIH) is a hereditaryrecessive disorder associated with deficiency of the alpha-L-iduronidaseenzyme, which is involved in the degradation of glycosaminoglycans(GAGs), or mucopolysaccharides. The accumulation of partially degradedGAGs causes interference with cell, tissue, and organ function. Theclinical features of Hurler syndrome include coarse facies, cornealclouding, mental retardation, hernias, dysostosis multiplex andhepatosplenomegaly. Children with Hurler syndrome appear normal at birthand develop the characteristic appearance over the first years of life[101]. In a group of patients of European descent with MPS I it wasestablished that the 2 most common mutations are the nonsense mutationsW402X and Q70X, which together account for approximately 70% of mutantalleles [102].

To evaluate the read-through efficiency induced by aminoglycosides, themost abundant nonsense mutations as underline causes of the abovediseases were selected. These included R3381X for DMD, R3X and R245X forUSH1, G542X and W1282X for CF, Q70X and W402X for Hurler Syndrome.

DNA fragments derived from the subsequent genes, including the testedstop mutation or the corresponding wild-type codon, and four upstreamand downstream flanking codons (except for p.R3X constructs, in whichthere are only two upstream PCDH15-flanking codons) were cloned intop2Luc plasmid between renilla and firefly luciferase genes as describedpreviously [90]. The formed plasmids, as well as the original p2Lucplasmid containing a TGA C nonsense mutation, were transcribed andtranslated in vitro and the stop codon suppression efficiency wascalculated as described previously [90].

The main advantage of this in vitro system, over the in vitro assay usedabove (see footnote of Table 20) is that in addition to rapid andprecise estimation of the read-through activity, it is also functionalassay wherein the assay deciphers the enzymatic activity of thetranslated luciferase proteins. In the previously used in vitro reportersystem, the readthrough activity was estimated by protein length only,while the activity of the produced full-length protein was not tested atall. Thus, the main advantage of this dual-luciferase reporter system isthe fact that misincorporation of amino acids that would affect theenzymatic activities of the luciferase proteins is also taken underconsideration. Using the dual luciferase reporter system, in vitrosuppression levels induced by Compound 37, Compound 3, and paromomycin,for each of the eight nonsense mutation carrying constructs were tested,and the results are presented in FIG. 5 and Table 21 below.

For the rat-PGM-β-Gal in vitro readthrough system, the pDB series ofreporter plasmids harbor the following elements under SP6 promotercontrol: an open reading frame (ORF) derived from the 5′ end of the ratPGM gene, which encodes a 25-kDa polypeptide; another small ORF,encoding the 10-kDa alpha complementation region of β-galactosidase, andterminated by tandem, in-frame stop codons (UAA UAG); located betweenthe two ORFs is a DNA fragment derived from PCDH15 cDNA, including thetested stop mutation or the corresponding wild-type codon, and sixupstream and downstream flanking codons (except for p.R3X constructs, inwhich there are only two upstream PCDH15-flanking codons). The fragmentswere created by annealing the following couples of complementaryoligonucleotides:

p.R3X: (SEQ ID NO: 3) 5′-GATCCATGTTTC/TGACAGTTTTATCTCTGGACA-3′; and(SEQ ID NO: 4) 5′-AGCTTGTCCAGAGATAAAACTGTCG/AAAACATG-3′; p.R245X:(SEQ ID NO: 5) 5′-GATCCCAAAATCTGAATGAGAGGC/TGAACAACCAC CACCACCCTCGCA-3′;and (SEQ ID NO: 6) 5′-AGCTTGCGAGGGTGGTGGTGGTTGTTCG/ACCTCTCATTCAGATTTTGG-3.

Usher Syndrome (PCDH15):

p.R3X: (SEQ ID NO: 7) 5′-TCGACATGTTTT/CGACCAGTTTTATCTCTGGACAGAGCT-3′;and (SEQ ID NO: 8) 5′-CTGTCCAGAGATAAAACTGT/GCAAAACATGGATCG-3′ p.R245X:(SEQ ID NO: 9) 5′-TCGACAAAATCTGAATGAGAGGT/CGAACAACCACCACCACCCTCGAGCT-3′; and (SEQ ID NO: 10)5′-CGAGGGTGGTGGTGGTTGTTCG/ACCTCTCATTCAGATTTTG-3′.

Cystic Fibrosis (CFTR):

p.G542X: (SEQ ID NO: 11) 5′-TCGACCAATATAGTTCTTT/GGAGAAGGTGGAATCGAGCT-3′; and (SEQ ID NO: 12) 5′-CGATTCCACCTTCTCA/GAAGAACTATATTGG-3′;p.W1282X: (SEQ ID NO: 13) 5′-TCGACAACTTTGCAACAGTGA/GAGGAAAGCCTTTGAGCT-3′; and (SEQ ID NO: 14)5′-CAAAGGCTTTCCTT/CCACTGTTGCAAAGTTG-3′.

Duchenne Muscular Dystrophy (Dystrophin):

p.R3381X: (SEQ ID NO: 15)5′-TCGACAAAAAACAAATTTTGA/CACCAAAAGGTATGAGCT-3′; and (SEQ ID NO: 16)5′-CATACCTTTTGGTT/GCAAAATTTGTTTTTTG-3′.

Hurler Syndrome (IDUA):

p.Q70X: (SEQ ID NO: 17) 5′-TCGACCCTCAGCTGGGACT/CAGCAGCTCAACCTCGAGCT-3′;and (SEQ ID NO: 18) 5′-CGAGGTTGAGCTGCTA/GGTCCCAGCTGAGG-3′; p.W402X:(SEQ ID NO: 19) 5′-TCGACTGAGGAGCAGCTCTGA/GGCCGAAGTGTCGGAGCT-3′; and(SEQ ID NO: 20) 5′-CCGACACTTCGGCT/CCAGAGCTGCTCCTCAG-3′.

The fragments were inserted into the BamHI and HindIII restriction sitesof the pDB read-through construct [6]. Reporter plasmids harboring eachof the four nonsense mutations and the corresponding wt alleles were invitro transcribed and translated using the TNT SP6 Coupled Reticulocytelysate System (Promega) in the presence of [³⁵S]-methionine andincreasing concentrations of aminoglycosides as described previously[6]. Reaction products were separated by electroporation on a 12.5%SDS-polyacrylamide gel, which was dried and subjected to PhosphorImageranalysis. The degree of suppression was calculated as the relativeproportion of the 35-kDa product out of total reaction products (35-kDaand 25-kDa polypeptides). The results are averages of at least threeindependent experiments.

For the dual luciferase in vitro transcription/translation assay, DNAfragments derived from PCDH15, CFTR, Dystrophin and IDUA cDNAs,including the tested stop mutation or the corresponding wild-type codon,and four upstream and downstream flanking codons (except for p.R3Xconstructs, in which there are only two upstream PCDH15-flanking codons)were created by annealing the couples of complementary oligonucleotidespresented above.

Fragments were inserted into the SalI and SacI restriction sites of thep2Luc plasmid between renilla and firefly luciferase genes [90]. Theafforded plasmids, as well as the original p2Luc plasmid containing aTGA C nonsense mutation, were transcribed and translated using the TNTReticulocyte Lysate Quick Coupled Transcription/Translation System(Promega™). Each transcription/translation reaction (10 μl, totalvolume) contained 8 μl TNT T7 Quick Master Mix, 0.2 μl methionine (1mM), 0.8 μl DNA template (2 μg/μl), 1 μl of either water (blank) or 1 μlsolution of increasing concentrations of the tested aminoglycosides inwater. Luciferase activity was determined following 90 minutesincubation at 30° C. using the Dual Luciferase Reporter Assay System(Promega™). The total luminescence produced by each one of the proteinswas measured separately using the FL×800 multidetection plate reader(Bio-Tek) and the stop codon suppression efficiency was calculated asdescribed previously [90]. The results are averages of at least threeindependent experiments.

For the, suppression of nonsense mutations by novel compounds was testedex vivo using a dual luciferase reporter plasmid ex vivo readthroughassay [90]. To generate the p2luc plasmid harboring the R3X nonsensemutation of the PCDH15 gene, the following oligonucleotides:5′-GATCCACAGAAGATGTTTTGACAGTTTTATCTCTGGACAGAGCT-3′ (SEQ ID NO: 21) and5′-CTGTCCAGAGATAAAACTGTCAAAACATCTTCTGTG-3′ (SEQ ID NO: 22) were annealedto each other and inserted into to polylinker sequence of the p2lucvector. The reporter plasmid p2luc-R3X was transfected to cos-7 cellswith Lipofectamine 2000 (Invitrogen) and addition of tested compoundswas performed 5 hours post transfection. Luciferase activity wasdetermined after 24 hours of incubation, using the Dual LuciferaseReporter Assay System (Promega) and stop codon readthrough wascalculated as described previously [90].

FIGS. 5A-H present the results of the in vitro stop codon suppressionlevels assays, induced by gentamicin (marked by black squares),paromomycin (marked by while squares), Compound 37 (marked by blackcircles) and Compound 3 (marked by white circles) in various nonsensemutation context constructs, p2luc (FIG. 5A), R3381X (Duchenne MuscularDystrophy) (FIG. 5B), R3X (FIG. 5C), R245X (Usher Syndrome) (FIG. 5D),G542X (FIG. 5E), W1282X (Cystic Fibrosis) (FIG. 5F), Q70X (FIG. 5G) andW402X (Hurler Syndrome) (FIG. 5H), and the suppression level wascalculated as the relation between the firefly and the renillaluciferases luminescence of the mutant and of the wild type vectors.

Table 21 presents the maximum in vitro suppression levels (by percent)of nonsense mutations from a panel of genetic diseases, which werederived from the data presented in FIGS. 5A-H. The compounds were testedin concentrations at which maximum suppression level was achieved.

TABLE 21 Tested Conc. Maximum in vitro suppression (%) compound (μM)UGAC R3X R245X G542X W1282X R3381X Q70X W402X 1 490 1.1 ± 0.1 0.6 ± 0.1ND ND ND ND ND ND NB56 878 7.1 ± 0.8 7.4 ± 0.7 1.4 ± 0.2 0.30 ± 0.090.57 ± 0.15 1.7 ± 0.3 3.5 ± 1.2 11.3 ± 1.5  gentamicin 46 17.7 ± 2.9 14.0 ± 1.0  0.90 ± 0.28 2.8 ± 0.7 1.4 ± 0.5 3.5 ± 0.7 1.5 ± 0.4 7.2 +0.5 paromomycin 42 7.8 ± 2.2 9.4 ± 0.5 0.71 ± 0.32 0.74 ± 0.08 1.2 ± 0.22.4 ± 0.3 2.2 ± 0.5 3.3 ± 0.1 Compound 3 107 4.8 ± 0.8 2.9 ± 0.9 0.98 ±0.30 0.63 ± 0.29 1.6 ± 0.4 1.8 ± 0.2 2.0 ± 0.4 3.2 ± 0.4 Compound 37 4618.4 ± 1.6  29.2 ± 3.1  4.9 ± 0.5 5.1 ± 0.6 3.3 ± 0.4 7.8 ± 0.8 10.6 ±1.1  21.9 ± 4.4 

As can be seen in Table 23, the measured half-maximal inhibitoryconcentration (IC₅₀) values show that all of the tested aminoglycosidesinhibit translation both in prokaryotes and eukaryotes, although, forall the compounds, there is significant preference for inhibitingtranslation in prokaryotes versus eukaryotes.

As can further be seen in Table 23, there is a noteworthy exception tothis trend in the case of Compound 37 and Compound 3. While bothgentamicin and paromomycin inhibit prokaryotic translation by about treeorder of magnitude preference than in eukaryotes, this difference inCompound 37 and Compound 3 drops to about two-order of magnitude.Furthermore, the efficacy by which both Compound 37 and Compound 3inhibit prokaryotic ribosome is significantly lower than that ofgentamicin and paromomycin. These data are in accord with the observedantibacterial data of this set of compounds, presented in Table 23.

Thus, while both gentamicin and paromomycin showed significantantibacterial activities against Escherichia coli (R477-100) with theminimal inhibitory concentrations (MIC) of 6 and 22 μM, respectively,both Compound 37 and Compound 3 exhibited lack of significantantibacterial activity (MIC values of 384 and 790 μM, respectively).Similar correlation between prokaryotic antitranslational activity andMIC values in E. coli has been reported previously [103].

In contrast to prokaryotic translation, both Compound 37 and Compound 3inhibit eukaryotic translation with higher efficacy than gentamicin andparomomycin. Compound 37 (IC₅₀=24 μM) is about 3-fold more efficientthan gentamicin (IC₅₀=60 μM) and paromomycin (IC₅₀=58 μM); Compound 3(IC₅₀=31 μM) is about 2-fold more efficient than these two drugs (see,Table 23). These collective results indicate that Compound 37 andCompound 3 are more specific for eukaryotic ribosomes than gentamicinand paromomycin. Furthermore, the specificity of Compound 37 tocytoplasmic ribosome is slightly greater than that of Compound 3.

Thus, the increased specificity observed together with the greateraffinity of Compound 37 versus Compound 3 to the cytoplasmic A siteshould be the basis for the observed improved suppression efficiency ofthe Compound 37 over that of Compound 3.

CONCLUSIONS

Compound 37, an exemplary compound according to the present embodiments,shows significantly improved stop codon readthrough activity andtoxicity. DNA fragments derived from the mutant PCDH15, CFTR, Dystrophinand IDUA genes carrying nonsense stop mutations and represent underlyingcauses for the genetic diseases USH1, CF, DMD and Hurler syndrome,respectively, were used to evaluate stop codon suppression ability ofCompound 37 relative to that of gentamicin and paromomycin in vitro. Inall mutations tested Compound 37 exhibited superior suppression activityto that of gentamicin and paromomycin. The efficiency of Compound 37over that of all other known aminoglycosides was greater for thesuppression of different stop codons (UGA and UAG), different flankingsequences surrounding the stop codons, and different identities of thefourth base immediately followed to the stop codon (C, A, and G). Incultured cell lines (COS-7 cells) Compound 37 exhibited maximumsuppression level of 5.25% of the R3X nonsense mutation, which was 3.2-,2.8- and 1.9-fold greater than that of paromomycin and gentamicin,respectively. Furthermore, Compound 37 has significantly reduced celltoxicity and acute toxicity in mice in comparison to paromomycin andgentamicin. The accumulative data indicates that Compound 37 may be moreefficiently used for suppression therapeutic purposes.

Example 4 Chemical-Enzymatic Attachment of an N-1-AHB Moiety toAminoglycoside Derivatives

Since the regioselective attachment of the AHB moiety on theaminoglycoside structure frequently requires long protection schemes [5]and the efficiency of a particular strategy is generally dependent onthe structure of the parent aminoglycoside, the present inventors haveused a shorter enzymatic approach as an alternative method. In thiscontext, the present inventors reported [104] that the biosynthesis ofButirosin B from Ribostamycin involves two sequential enzymatic steps,as illustrated in Scheme 11 below.

As can be seen in Scheme 11, the AHB moiety is first transferred fromthe acyl carrier protein Btrl to the precursor aminoglycosideribostamycin as a γ-glutamylated dipeptide by the acyltransferase enzymeBtrH to yield γ-L-Glu-butirosin B; the protective γ-glutamyl group isthen cleaved by the BtrG enzyme via an intramolecular transamidationmechanism. The application of this method to the combinedchemical-enzymatic production of a variety of the novel N-1-AHB-bearingaminoglycoside derivative compounds according to the present embodimentswas particularly suitable because the recombinant BtrH and BtrG enzymesare easily accessible as N-terminally His₆-tagged proteins [104], andthat the native acyl donor γ-L-Glu-AHB-S-Btrl (see, Scheme 11), which israther difficult to produce in large quantities, can be very efficientlyreplaced by the synthetic N-acetylcysteamine thioester γ-L-Glu-AHB-SNAC(see, Scheme 11) [105]. In addition, preliminary tests of this system'stolerance for alternative aminoglycoside acceptors indicated that, inaddition to ribostamycin, related native aminoglycosides such asneomycin and paromomycin could also be efficiently converted to thecorresponding N-1-AHB derivatives [105].

Accordingly, an efficient chemoenzymatic method for the preparation of avide variety of 2-DOS-containing aminoglycosides with the valuable AHBpharmacophore according to the present embodiments, has been developed,using the BtrH/BtrG catalytic system with the synthetic acyl donorγ-L-Glu-AHB-SNAC. Since the compound presented herein are polycationicand thus are water soluble substances, the addition of the AHB sidechain is done in the final steps of the synthesis, and as such, thepresented method significantly shortens and simplifies the chemicalstrategies that necessitate long protection schemes for this purpose.The observed broad substrate tolerance of the BtrH enzyme for thesynthetic substances tested so far, along with the easy accessibility ofthe recombinant BtrH and BtrG enzymes also make this method attractivefor high throughput synthesis of a library of 2-DOS-containingaminoglycosides to discover valuable hits with potent biomedicalrelevance.

Introduction of an AHB Moiety—A General Procedure:

Following is a general procedure for the attachment of an AHB moiety toa reactive amino group in an aminoglycoside derivative compoundaccording to the present embodiments, as illustrated in Scheme 12 below.

To the reaction mixture (total volume of 50 μL) containing HEPES buffer(50 mM, pH 7.9), synthetic acyl donor γ-L-Glu-AHB-SNAC (5 mM) and aprecursor of a compound for AHB attachment (1.2 mM), according to thepresent embodiment, is added the purified BtrH enzyme (125 g) and themixture is incubated at 20° C. for 6 hours. The protein is removed byaddition of 20 μl chloroform followed by vortexing and centrifugation(13,000 rpm, 5 minutes). The clear aqueous layer is taken for the nextenzymatic step without further purification.

A small aliquot (about 1 μl) of this solution is taken for LC-ESI-MS/MSanalysis to determine the percentage of conversion of the aminoglycosidesubstrate to the desired product.

To the aqueous layer from the previous step is added the purified BtrGenzyme (18 g) and the mixture is incubated at 20° C. for 24 hours. Theprotein is removed as above and the aqueous layer is taken forLC-ESI-MS/MS analysis using an Agilent HP1100 HPLC system coupled to aThermo-Finnigan LCQ ion-trap mass spectrometer equipped with anelectrospray ionization (ESI) source.

After enzymatic reactions the samples are separated on a 2.0×250 mm Luna5μ C18(2) column (Phenomenex) by the following gradient at a flow rateof 0.3 ml/min and column temperature of 40° C.: 0-20 minutes 10%-50% B,20-21 minutes 50%-10% B, 21-25 minutes 10% B (buffer A: 0.1%pentafluoropropionic acid (PFPA) in H₂O; buffer B: 0.1% PFPA in MeCN).Mass spectra were acquired from 250 to 1000 Da. MS/MS is carried out ontarget ions with 20% relative collision energy (helium as collisiongas).

Preparative scale reactions are performed as above in a total volume of10-15 ml, and with the addition of 1.5 mg BtrH and 1.0 mg BtrG. Theincubation time for both enzymatic steps is also extended to 24 hours.The aqueous layer, after removal of BtrG, is loaded onto Dowex 50W (NH₄⁺ form) 15×80 mm ion-exchange column. The column is washed with water(50 mL) followed by elution with 1% NH₄OH in water. Fractions containingproduct, detected by TLC using CH₂Cl₂/MeOH/H₂O/MeNH₂ (33% solution inEtOH, 10:15:6:15), are combined and evaporated to dryness. The residueis dissolved in water, the pH is adjusted to 3.5 by H₂SO₄ (0.05 M) andlyophilized to afford the sulfate salt which is used for all thespectral analyses.

Chemical-Enzymatic Preparation of Compound 37:

Compound 37 (also referred to herein as NB54) was prepared from Compound3 (also referred to herein as NB30) with full preservation of N-1regioselectivity by BtrH with according to the General Procedureprovided hereinabove, as illustrated in Scheme 13 and compared to thechemically synthesized sample which was prepared as describedhereinabove.

The recombinant BtrH and BtrG enzymes were isolated as homogeneousN-terminally His₆-tagged proteins according to the previously reportedprocedures [104]. The synthetic acyl donor γ-L-Glu-AHB-SNAC wassynthesized as previously described [105]. Compound 3 was synthesized asdescribed hereinabove.

FIGS. 8A-D present a comparison of ¹H NMR spectra (FIGS. 8A and 8B) and¹³C NMR spectra (FIGS. 8C and D) of Compound 37 prepared by thechemo-enzymatic procedure presented herein (FIGS. 8A and 8C) and by thechemical procedure presented herein (FIGS. 8B and 8D), whereas the “*”denotes unidentified impurities.

FIGS. 9A-C present a comparison of 2D-COSY spectra of Compound 37prepared by chemical (FIG. 9A) and chemoenzymatic (FIG. 9B) procedurespresented herein, with that of the Compound 3 (FIG. 9C), whereas thedashed lines show correlations between 2-Hax and 2-Heq protons with 1-Hand 3-H protons of the 2-DOS ring, highlighting strong downfield shiftof the 1-H proton in Compound 37 versus 1-H proton in the parentcompound Compound 3.

As can be seen in FIGS. 8 and 9, all the 1D and 2D NMR data of theenzymatic product, under the same pH and counterion conditions, areidentical to that of the synthetic compound. Furthermore, preliminarytests of Compound 37 for in vitro readthrough activity of the TGA stopcodon, demonstrated that it exhibits significantly higher stop codonread-through activity than its parent Compound 3 and the natural drugparomomycin.

In conclusion, it has been demonstrated herein that an efficientchemoenzymatic process for the preparation of a vide variety of2-DOS-containing aminoglycosides with the valuable AHB pharmacophore byusing the BtrH/BtrG catalytic system with the synthetic acyl donorγ-L-Glu-AHB-SNAC, can afford the compounds according to the presentembodiments.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

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What is claimed is:
 1. A method of inducing readthrough of a prematurestop codon mutation in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of acompound having a general Formula I:

or a pharmaceutically acceptable salt thereof, wherein: each of R₁, R₂and R₃ is independently a monosaccharide moiety, halide, hydroxyl, amineor an oligosaccharide moiety, at least one of R₁, R₂ and R₃ being amonosaccharide moiety having the general Formula II:

wherein each of R₆, R₇ and R₈ is independently selected from the groupconsisting of hydroxyl and amine, or R₁ being an oligosaccharide moiety,whereas when R₂ is said monosaccharide moiety having said Formula II, R₆is amine; X is oxygen or sulfur; R₄ is (S)-4-amino-2-hydroxybutyryl(AHB); R₅ is hydroxyl; Y is hydrogen or alkyl; and each of the dashedlines indicates independently an R configuration or an S configuration.2. The method of claim 1, wherein said compound is:


3. The method of claim 1, wherein X is oxygen.
 4. The method of claim 1,wherein Y is hydrogen.
 5. The method of claim 1, wherein at least one ofR₁, R₂ and R₃ is a disaccharide moiety.
 6. The method of claim 1,wherein Y is alkyl.
 7. The method of claim 1, wherein said prematurestop codon mutation is associated with a genetic disorder, the methodbeing for treating said genetic disorder in the subject.
 8. The methodof claim 7, wherein said genetic disorder is selected from the groupconsisting of cystic fibrosis (CF), Duchenne muscular dystrophy (DMD),ataxia-telangiectasia, Hurler syndrome, hemophilia A, hemophilia B,Usher syndrome, Tay-Sachs Becker muscular dystrophy (BMD), Congenitalmuscular dystrophy (CMD), Factor VII deficiency, Familial atrialfibrillation, Hailey-Hailey disease, McArdle disease,Mucopolysaccharidosis, Nephropathic cystinosis, Polycystic kidneydisease, Rett syndrome, Spinal muscular atrophy (SMA), X-linkednephrogenic diabetes insipidus (XNDI) and X-linked retinitis pigmentosa.9. A method inducing readthrough of a premature stop codon mutation in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a compound having ageneral Formula I:

or a pharmaceutically acceptable salt thereof, wherein: each of R₁, R₂and R₃ is independently a monosaccharide moiety, hydroxyl, amine or anoligosaccharide moiety, and at least one of R₁, R₂ and R₃ being amonosaccharide moiety having the general Formula II:

wherein each of R₆, R₇ and R₈ is independently selected from the groupconsisting of hydroxyl and amine, or R₁ being an oligosaccharide; X isoxygen or sulfur; R₄ is hydrogen; R₅ is hydroxyl; Y is hydrogen; andeach of the dashed line indicates independently an R configuration or anS configuration, with the proviso that said compound is not selectedfrom the group consisting of

and paromomycin.
 10. The method of claim 9, wherein X is oxygen.
 11. Themethod of claim 9, wherein said compound is:


12. A method of inducing readthrough of a premature stop codon mutationin a subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a compound having ageneral Formula I:

or a pharmaceutically acceptable salt thereof, wherein: each of R₁, R₂and R₃ is independently a monosaccharide moiety, halide, hydroxyl, amineor an oligosaccharide moiety, X is oxygen or sulfur; R₄ is hydrogen or(S)-4-amino-2-hydroxybutyryl (AHB); R₅ is hydroxyl or amine; Y is alkyl;and each of the dashed line indicates independently an R configurationor an S configuration, and wherein: at least one of R₁, R₂ and R₃ isselected from a monosaccharide moiety and an oligosaccharide moiety;and/or R₄ is (S)-4-amino-2-hydroxybutyryl (AHB), with the proviso thatsaid compound is not G-418.
 13. The method of claim 12, wherein X isoxygen.
 14. The method of claim 12, wherein R₅ is hydroxyl.
 15. Themethod of claim 12, wherein at least one of R₁, R₂ and R₃ is amonosaccharide moiety.
 16. The method of claim 15, wherein R₄ is AHB.17. The method of claim 12, wherein said compound is:


18. The method of claim 12, wherein said premature stop codon mutationis associated with a genetic disorder, the method being for treatingsaid genetic disorder in the subject.
 19. The method of claim 18,wherein said genetic disorder is selected from the group consisting ofcystic fibrosis (CF), Duchenne muscular dystrophy (DMD),ataxia-telangiectasia, Hurler syndrome, hemophilia A, hemophilia B,Usher syndrome, Tay-Sachs Becker muscular dystrophy (BMD), Congenitalmuscular dystrophy (CMD), Factor VII deficiency, Familial atrialfibrillation, Hailey-Hailey disease, McArdle disease,Mucopolysaccharidosis, Nephropathic cystinosis, Polycystic kidneydisease, Rett syndrome, Spinal muscular atrophy (SMA), X-linkednephrogenic diabetes insipidus (XNDI) and X-linked retinitis pigmentosa.20. A method of inducing readthrough of a premature stop codon mutationin a subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a compound having ageneral Formula I:

or a pharmaceutically acceptable salt thereof, wherein: each of R₁, R₂and R₃ is independently a monosaccharide moiety, halide, hydroxyl, amineor an oligosaccharide moiety, X is oxygen or sulfur; R₄ is(S)-4-amino-2-hydroxybutyryl (AHB); R₅ is hydroxyl or amine; Y is alkyl;and each of the dashed line indicates independently an R configurationor an S configuration.
 21. The method of claim 20, wherein X is oxygen.22. The method of claim 20, wherein R₅ is hydroxyl.
 23. The method ofclaim 20, wherein at least one of R₁, R₂ and R₃ is a monosaccharidemoiety.
 24. The method of claim 20, wherein said premature stop codonmutation is associated with a genetic disorder, the method being fortreating said genetic disorder in the subject.
 25. The method of claim24, wherein said genetic disorder is selected from the group consistingof cystic fibrosis (CF), Duchenne muscular dystrophy (DMD),ataxia-telangiectasia, Hurler syndrome, hemophilia A, hemophilia B,Usher syndrome, Tay-Sachs Becker muscular dystrophy (BMD), Congenitalmuscular dystrophy (CMD), Factor VII deficiency, Familial atrialfibrillation, Hailey-Hailey disease, McArdle disease,Mucopolysaccharidosis, Nephropathic cystinosis, Polycystic kidneydisease, Rett syndrome, Spinal muscular atrophy (SMA), X-linkednephrogenic diabetes insipidus (XNDI) and X-linked retinitis pigmentosa.